Method for altering degradation of engineered protein in plant cells

ABSTRACT

A method of altering degradation of heterologous proteins in transgenic plants has now been found that utilizes ER-localizing proteins of plant viruses as part of a fusion protein. An engineered fusion protein is protected from degradation by a viral ER-localizing protein, and made more susceptible to degradation by certain mutant viral proteins that fail to localize to the ER.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U. S. Provisional PatentApplication Serial No. 60/218,504, filed Jul. 15, 2000.

TECHNICAL FIELD OF INVENTION

[0002] This invention relates to a method of altering the rate ofdegradation of proteins in plant cells.

BACKGROUND OF THE INVENTION

[0003] Intracellular protein concentration is influenced by manyfactors, including the rates of transcription, translation, anddegradation. When cells are engineered to express a protein from atransgene, robust and stable expression may be desired. In othercircumstances, limited accumulation of a protein from a transgene may bedesired. Being able to modulate an engineered protein's rate ofdegradation has numerous applications.

[0004] One advantage of being able to manipulate a protein's degradationrate is to increase its intracellular concentration to study itsfunction. After translation, protein levels are controlled by proteaseactivity (Vierstra, 1996) which can limit the accumulation of proteinsunder study to levels that prevent their biochemical characterization.Another advantage to increasing a selected protein's intracellularconcentration is that it may enhance the accumulation of foreignproteins with beneficial traits in transgenic plants (Vierstra, 1996).As a contrast, there may also be advantages to enhancing a protein'sdegradation. The identification of sequences that lead to fasterdegradation of proteins will benefit researchers interested inrepressing accumulation of unwanted endogenous proteins that interferewith important agronomic processes (Vierstra, 1996). Interest in methodsto regulate protein accumulation is reflected by the approaches thathave been previously reported. One method includes modifying the primarysequence to remove domains conferring instability, and another method isto inhibit proteases (reviewed in Vierstra, 1996.) In another instance,ubiquitin, a stable protein, was fused to a poorly expressed protein toenhance the expression of the latter (Eker et al. 1989).

[0005] Non-host proteins are produced in viral-infected plants. Duringtobacco mosaic virus (TMV) infection, two such proteins are the 126 kDaprotein and the 183 kDa protein, a read-through product containing the126 kDa protein sequence. Description of these proteins in the prior artindicate that they play a role in replication. Approximately 10% of the126 kDa protein heterodimerizes with essentially all of the 183 kDaprotein in the plant cell, even though the 183 kDa protein alone iscapable of replicating the virus in infected cells (Watanabe et al.,1999; Lewandowski and Dawson, 2000). Both proteins are reportedlyrequired for efficient TMV replication in vivo (Osman and Buck, 1996;Watanabe et al., 1999). In fact, the 126 kDa/183 kDa proteins were foundwith other TMV and host plant factors in the viral replication complex(Heinlein et al., 1998). Additionally, the 126 kDa/183 kDa proteins haveputative methyltransferase and helicase domains. Furthermore, the 183kDa protein contains a carboxy terminal domain required forRNA-dependent RNA polymerase activity.

[0006] Although the role of the 126 kDa protein and/or the 183 kDaprotein of TMV is thought in the prior art to be replication, itsintracellular localization was unknown. Mas and Beachy (1999) observedthat the 126 kDa protein of TMV co-localizes with viral RNA insubcellular bodies and with luminal binding protein (BiP), anendoplasmic reticulum (ER)-specific protein, in infected plants.Although these observations suggested to Mas and Beachy that the 126 kDaprotein and/or the 183 kDa protein of TMV localizes to the ER, thelocalization signal of the proteins was not identified.

[0007] Comparing the 126 kDa protein and/or the 183 kDa protein of TMVto another species suggested in the prior art that the proteins maylocalize to the ER. Brome mosaic virus BMV), a virus related to Tobaccomosaic virus (TMV), possesses a protein believed to be homologous infunction to the 126 kDa protein of TMV, although its overall sequenceidentity with the TMV protein is 13%. Previous publications determinedthat the BMV 1a protein localized to the ER during infection of barleycells and that, in the absence of other viral proteins, it localized tothe ER in yeast (Restrepo-Hartwig and Ahlquist 1999). Therefore, the BMV1a protein, like its putative TMV homolog, may localize to specificsubcellular locations. In additional to localizing to the endoplasmicreticulum in yeast, the 1a protein also stabilized viral RNA (Sullivanand Ahlquist, 1999) and decreased the viral RNA translation (Janda andAhlquist, 1998).

[0008] The post-translational regulation of the 126 kDa protein and/orthe 183 kDa protein of TMV has also been studied in the prior art, butonly with ambiguous results. Previous reports indicated that 26Sproteasome inhibitors had no significant effect on 126 kDa or 183 kDaprotein accumulation in plant cell suspensions infected with TMV(Reichel and Beachy, 2000). In late stages of TMV infection, what littleeffect occurred indicated that the protein was more susceptible todegradation in the presence of the 26S proteasome inhibitor. From theseresults it appeared that induction of 26S proteasome activity had nosignificant influence on the degradation of the 126 kDa protein.Importantly, the ability of the 126 kDa protein to stabilize itsexpression in the absence of other viral proteins was not tested inthese studies by Reichel and Beachy.

[0009] There is a desire and a need in agronomic biotechnology tomodulate the expression level of engineered proteins. Expression incells engineered to express a protein from a transgene may be robust andstable; in other circumstances, limited accumulation of a protein from atransgene may be desired. To fulfill that need, we have developed aubiquitin-fusion—independent system in which the degradation—and hencethe protein level—of an engineered protein can be modulated in plantcells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 is a partial protein sequence alignment (amino acids361-370 of SEQ ID NO:2 and SEQ ID NO:4) of the TMV 126/183 kDa proteinand its functional analogs from Sindbis—like plant viruses. Theconserved “WFP” motif is boxed and the amino acids in bold type areidentical to amino acids 365-367 of SEQ ID NO:2 and SEQ ID NO:4. Theunderlined letters (serine 361 and lysine 368) show the amino acids inthe TMV U1 strain that are different from that of the M^(IC) strain.AMV: alfalfa mosaic virus (SEQ ID NO:9); BMV: brome mosaic virus (SEQ IDNO:10); CiLRV: citrus leaf rugose virus (SEQ ID NO:11); CMV: cucumbermosaic virus (SEQ ID NO:12); SHMV: sunn-hemp mosaic virus (SEQ IDNO:13); TMV: tobacco mosaic virus U1 strain (SEQ ID NO:14); TRV: tobaccorattle virus (SEQ ID NO:15); and TVCV: turnip vein clearing virus (SEQID NO:16).

[0011]FIG. 2A depicts the genome organization of TMV. Three open readingframes (ORFs) which encode the 126 kDa protein (1-3348 of SEQ ID NO:1)and the read-through 183 kDa protein (1-4831 of SEQ ID NO:3), themovement protein (horizontal stripes), and the coat protein (dotted). Ablack arrowhead indicates the location of the leaky amber stop codon(UAG) within the replicase ORF. The methyltransferase domain of the126/183 kDa protein is represented with vertical stripes, beginning atnucleotide 142 of SEQ ID NO:1 and ending at nucleotide 900 of SEQ IDNO:1. The helicase domain of the 126/183 kDa protein is represented withdiamonds, beginning at nucleotide 2362 of SEQ ID NO:1 and ending atnucleotide 3249 of SEQ ID NO:1. GDD (white) is a motif present in viralRNA-dependent RNA polymerase. Domains I and II of the 126/183 kDaprotein each have 4 amino acid mutations that were identified to controlthe phenotype difference between the TMV U1 strain, which causes severesymptoms, and the cloned Masked strain of TMV (M^(IC)), which causesmild symptoms. Nucleotide numbers in the figure refer to the entiregenome of TMV, Genbank Accession No. AF273221.

[0012]FIG. 2B depicts the different amino acids present within Domains Iand II of the 126/183 kDa protein and the resulting symptoms of theviruses. The following sequences were aligned: TMV-U1 (SEQ ID NO:17),the parental TMV-M^(IC) (SEQ ID NO:18), and the site-directed mutantviruses studied, TMV-M^(IC)2 (SEQ ID NO:19), TMV-WAP (SEQ ID NO:20), andTMV-WYP (SEQ ID NO:21). Among all sequences, the amino acids in 8positions (four mutations in each of two domains) were determined tovary: 325, 360, 367, 416, 587, 601, 668, and 747, referring to theentire genome of TMV, Genbank Accession No. AF273221.

[0013]FIG. 3A depicts the lesion response (pictorially white spots) on aN. tabacum Xanthi “NN” leaf challenged with WFP and WYP viruses. Eachhalf of the leaf was inoculated with either the WFP or WYP virus and theplant grown at 24° C. for ten days. The side of the leaf inoculated withthe WFP virus resulted in a slightly larger lesions than the side of theleaf infected with the WYP virus.

[0014]FIG. 3B depicts the lesion response (pictorially white spots) on aN. tabacum Xanthi “NN” leaf challenged with WFP and WYP viruses. Eachhalf of the leaf was inoculated with either the WFP or WYP virus and theplant grown at 32° C. for three days. The temperature was then decreasedto 24° C. for another seven days. The size of lesions on the WFPvirus—treated side of the leaf increased relative to the side of theleaf treated with the WYP virus.

[0015] FIGS. 4A-H depict immunolabeling experiments in N. tabacum BY-2protoplasts. All images for FIGS. 4A-H were captured by confocal laserscanning microscopy using a previously described procedure (Cheng, etal., 2000). Bar=20 μM. FIG. 4A depicts immunolabeling of the 126 kDaprotein in an N. tabacum BY-2 protoplast infected with the WFP virus.The pictorially light region indicates the presence and location of the126 kDa protein. FIG. 4B depicts immunolabeling of BiP in an N. tabacumBY-2 protoplast infected with the WFP virus. The pictorially lightregion indicates the presence and location of the BiP protein. FIG. 4Cdepicts immunolabeling of the 126 kDa protein in an N. tabacum BY-2protoplast infected with the WYP virus. The pictorially light regionindicates the presence and location of the 126 kDa protein. FIG. 4Ddepicts immunolabeling of BiP in an N. tabacum BY-2 protoplast infectedwith the WYP virus. The pictorially light region indicates the presenceand location of the BiP protein.

[0016]FIG. 4E depicts immunolabeling of the 126 kDa protein in an N.tabacum BY-2 protoplast infected with the M^(IC) virus. The pictoriallylight region indicates the presence and location of the 126 kDa protein.FIG. 4F depicts immunolabeling of BiP in an N. tabacum BY-2 protoplastinfected with the M^(IC) virus. The pictorially light region indicatesthe presence and location of the BiP protein. FIG. 4G depictsimmunolabeling of the 126 kDa protein in a mock-inoculated N. tabacumBY-2 protoplast. As expected, there is no detection of the 126 kDaprotein. FIG. 4H depicts immunolabeling of BiP in a mock-inoculated N.tabacum BY-2 protoplast. The pictorially light region indicates thepresence and location of the BiP protein that was not localized, unlikewhen 126 kDa protein from the WFP or M^(IC) virus was present.

[0017]FIG. 5A is a diagram of a portion of genetic constructs bombardedinto host leaves for transient expression. Open arrows depict theenhanced 35 S promoter; the dotted box represents nucleotides 1-3348 ofSEQ ID NO:1 (for construct 126F:GFP) that encodes the 126 kDa proteinfrom TMV; the box with diagonal stripes represents the DNA encoding forGFP (EGFP, Clontech Laboratories, Palo, Alto, Calif.); and the filledarrow represents the mRNA termination sequence. The bolded letter in thesequence depicted in the 126 kDa protein indicates the amino aciddifferences among the constructs. For construct 126Y:GFP, nucleotides1-3348 of SEQ ID NO:5 where nucleotides that encode amino acid 366 are“ata” were inserted, and for construct 126A:GFP, nucleotides 1-3348 ofSEQ ID NO:5 where nucleotides that encode amino acid 366 are “agc” wereinserted. Each genetic element with the exception of the nucleotidesencoding the 126 kDa protein from TMV originated in the expressionvector pRTL2 (Topfer, et al. 1987 and Restrepo-Hartwig, et al. 1990).

[0018]FIGS. 5B-5G depict transient expression of the WFP-containing 126kDa:GFP fusion proteins in N. tabacum (N.t) and N. benthamiana (N.b)leaves. White color on black background indicates the presence of fusedprotein. At 16 hours post-bombardment, the WFP-containing 126 kDa:GFPfusion construct bombarded onto N. tabacum leaves has similar expressionto that of the same construct inoculated on N. benthamiana leaves (FIGS.5B and 5C). This trend continues through the 44 hour and 8 day timepoints (FIGS. 5D and 5E and FIGS. 5F and 5G, respectively).

[0019]FIGS. 5H-5M depict transient expression of the WAP-containing 126kDa:GFP fusion proteins in N. tabacum (N.t) and N. benthamiana (N.b)leaves. White color on black background indicates the presence of fusedprotein. The WAP-containing 126 kDa:GFP fusion construct bombarded ontoN. benthamiana leaves has similar expression 16 hours post-bombardmentthan the same construct inoculated on N. tabacum leaves (FIGS. 5H and5I). At 44 hours post-bombardment, there is more 126 kDa:GFP fusionexpression on N. benthamiana leaves than at 16 hours, but far less 126kDa:GFP fusion expression on N. tabacum leaves than the previous timepoint (FIGS. 5J and 5K). By 8 days there is low expression of the 126kDa:GFP fusion expression on N. benthamiana leaves and no expression onN. tabacum leaves (FIGS. 5L and 5M).

[0020]FIGS. 5N-5S depict transient expression of the WYP containing 126kDa:GFP fusion proteins in N. tabacum (N.t) and N. benthamiana (N.b)leaves. White color on black background indicates the presence of fusedprotein. At 16 hours post-bombardment, there is similar expression ofthe WYP-containing 126 kDa:GFP fusion constructs in N. tabacum and N.benthamiana leaves (FIGS. 5N and 5O). At 44 hours post-bombardment, theexpression of the WYP-containing 126 kDa:GFP fusion constructs on N.tabacum and N. benthamiana leaves appears similar and low (FIGS. 5P and5Q). At 8 days post-bombardment, significant expression of theWYP-containing 126 kDa:GFP fusion constructs on N. benthamiana leavesremain, whereas there is little if any expression of the WYP-containing126 kDa:GFP fusion constructs on N. tabacum leaves (FIGS. 5R and 5S).

[0021]FIGS. 6A-6H depict the resulting expression of the 126F:GFP(WFP-containing construct), 126Y:GFP (WYP-containing construct), and126A:GFP (WAP-containing construct) in N. benthamiana protoplasts.Although fluorescent bodies are detected in all protoplastselectroporated with the fusion constructs (FIGS. 6A-6F), the size of thebodies is smaller in the protoplast electroporated with the 126A:GFPconstruct (FIG. 6A) than in the other protoplasts (FIGS. 6C and 6E) 7hours after electroporation. At 24 hours after electroporation, theprotoplasts expressing the 126F:GFP and 126Y:GFP constructs appear tohave fewer, but larger fluorescent bodies (FIGS. 6D and 6F). Theprotoplasts expressing free GFP form no punctate bodies even after 24hours (FIGS. 6G and 6H). Bar=10 μM.

[0022]FIG. 7A provides the quantities of large (>2 μM) fluorescentbodies per protoplast formed by the transiently expressed WFP-, WYP-, orWAP-containing fusion proteins in N. benthamiana protoplasts over time(means±SD). The bars with horizontal stripes represent the expression ofthe WFP-containing fusion construct. The bars with the vertical stripesrepresent the expression of the WYP-containing fusion construct. Thebars with diagonal stripes represent the expression of theWAP-containing fusion construct. The number of large bodies in N.benthamiana protoplasts transiently expressing WFP-, WYP-, orWAP-containing fusion proteins does not significantly differ amongtreatments at 16-36 hours. However, after 48 hours N. benthamianaprotoplasts transiently expressing the WFP-containing fusion proteinhave more large bodies than protoplasts transiently expressing the otherfusion proteins and the difference exists at the 72 and 96 hour timepoints as well.

[0023]FIG. 7B provides the quantities of small (<2 μM) fluorescentbodies formed by the transiently expressed WFP-, WYP-, or WAP-containingfusion proteins in N. benthamiana protoplasts over time (means±SD).Generally, within each treatment the amounts of small fluorescent bodiesdecrease with time. Although there is no significant difference betweentreatments at each time point, the N. benthamiana protoplasts expressingthe WYP-containing fusion protein appear to have a greater number ofsmall bodies than the other treatments until the 96 hour time point.

[0024]FIG. 7C provides the ratio of small (<2 μM) fluorescent bodies tolarge (>2 μM) fluorescent bodies formed by the transiently expressedWFP-, WYP-, or WAP-containing fusion proteins in N. benthamianaprotoplasts over time (means±SD). There does not appear to besignificant differences between treatments at every time point, but thesmallest ratio of small to large fluorescent bodies in N. benthamianaprotoplasts have WFP-containing fusion proteins at 48-96 hourspost-electroporation.

[0025]FIGS. 8A-8F depict the transient expression of WFP-containingfusion proteins in N. tabacum BY-2 protoplasts in the presence (FIGS.8B, 8D, and 8F) or absence (FIGS. 8A, 8C, and 8E) of a ubiquitin pathwayinhibitor, ALLN, over time (12, 24, and 48 hours). There is greaterWFP-containing fusion protein expression in protoplasts treated withALLN than without ALLN at every time point (compare FIG. 8A to FIG. 8B,FIG. 8C to FIG. 8D, and FIG. 8E to FIG. 8F). Bar=10 μM.

[0026]FIGS. 8G-8L depict the transient expression of WYP-containingfusion proteins in N. tabacum BY-2 protoplasts in the presence (FIGS.8H, 8J, and 8L) or absence (FIGS. 8G, 8I, and 8K) of a ubiquitin pathwayinhibitor, ALLN, over time (12, 24, and 48 hours). There is greaterWFP-containing fusion protein expression in protoplasts treated withALLN than without ALLN at every time point (compare FIG. 8G to FIG. 8H,FIG. 8I to FIG. 8J, and FIG. 8K to FIG. 8L). However, transientexpression in the absence of ALLN peaked 24 hours post-electroporationwith little expression at 48 hours post-electroporation. In BY-2protoplasts expressing the WYP-containing fusion protein and treatedwith ALLN, the number of small bodies decreased as the large bodiesincreased in size over time (FIG. 8H, 8J and 8K). Bar=10 μM.

[0027]FIGS. 8M-8R depict the transient expression of WAP-containingfusion proteins in N. tabacum BY-2 protoplasts in the presence (FIGS.8N, 8P, and 8R) or absence (FIGS. 8M, 8O, and 8Q) of a ubiquitin pathwayspecific inhibitor, ALLN, over time (12, 24, and 48 hours). At 12 and 24hours post-electroporation, BY-2 protoplasts in the presence of ALLNtransiently express more WAP-containing fusion protein than thetime-matched, -ALLN protoplasts (compare FIG. 8M to FIG. 8N and FIG. 8Oto FIG. 8P). Also, there was greater WAP-containing fusion proteinexpression in ALLN treated BY-2 protoplasts at 24 hours than at 12 hourspost-electroporation (FIG. 8N and FIG. 8P). However, at 48 hours, therewas no detectable WAP-containing fusion protein expression in BY-2protoplasts in the absence of ALLN (FIG. 8Q) and only very little, butaggregated, expression in the presence of ALLN (FIG. 8R). Bar=10 μM.

SUMMARY OF THE INVENTION

[0028] In one aspect, the invention is a method for decreasing thedegradation rate of an engineered protein of interest in a plant cellcomprising the steps a) constructing a vector comprising a nucleic acidfragment from position 1 to position 3348 of SEQ ID NO:1 fused to anucleotide sequence encoding a protein of interest, the vectorexpressible in said plant cell; and b) introducing and expressing thevector in the plant cell to form a fused protein, wherein thedegradation rate of the fused protein is less than the degradation rateof the engineered protein of interest in the plant cell or a plant cellof the same species. The vector may be integrated into the genome ofsaid plant cell. The invention is furthermore a plant cell transformedaccording to the above method and a plant generated from the transformedplant cell.

[0029] In another aspect, the invention is a method for decreasing thedegradation rate of an engineered protein of interest in a plant cellcomprising the steps a) constructing a vector comprising a nucleic acidfragment from position 1 to position 4831 of SEQ ID NO:3 fused to anucleotide sequence encoding a protein of interest, the vectorexpressible in said plant cell; and b) introducing and expressing thevector in the plant cell to form a fused protein, wherein thedegradation rate of the fused protein is less than the degradation rateof the engineered protein of interest in the plant cell or a plant cellof the same species. The vector may be integrated into the genome ofsaid plant cell. The invention is furthermore a plant cell transformedaccording to the above method and a plant generated from the transformedplant cell.

[0030] In another aspect, the invention is also a method for increasingthe degradation rate of an engineered protein of interest in a plantcell comprising the steps a) constructing a vector comprising a nucleicacid fragment from position 1 to position 3348 of SEQ ID NO:5 fused to anucleotide sequence encoding a protein of interest, the vectorexpressible in a plant cell; and b) introducing and expressing thevector in the plant cell to form a fused protein, wherein thedegradation rate of the fused protein is less than the degradation rateof the engineered protein of interest in the plant cell or a plant cellof the same species. Nucleotides at positions 1096-1098 of SEQ ID NO:5encode alanine or tyrosine. The vector may be integrated into the genomeof said plant cell. The invention is furthermore a plant celltransformed according to the above method and a plant generated from thetransformed plant cell.

[0031] In another aspect, the invention is also a method for increasingthe degradation rate of an engineered protein of interest in a plantcell comprising the steps a) constructing a vector comprising a nucleicacid fragment from position 1 to position 4831 of SEQ ID NO:7 fused to anucleotide sequence encoding a protein of interest, the vectorexpressible in a plant cell; and b) introducing and expressing thevector in the plant cell to form a fused protein, wherein thedegradation rate of the fused protein is less than the degradation rateof the engineered protein of interest in the plant cell or a plant cellof the same species. Nucleotides at positions 1096-1098 of SEQ ID NO:7encode alanine or tyrosine. The vector may be integrated into the genomeof said plant cell. The invention is furthermore a plant celltransformed according to the above method and a plant generated from thetransformed plant cell.

[0032] In another aspect, the invention is a purified nucleic acidcomprising a nucleic acid fragment from position 1 to position 3348 ofSEQ ID NO:1 fused to a DNA sequence encoding a protein of interest,wherein expression of said purified nucleic acid in a plant cell resultsin a fusion protein having increased stability when compared to thestability of said protein of interest engineered without fusionexpressed in a plant cell of the same species. The invention is also theresulting fusion protein comprising SEQ ID NO:2 encoded by the purifiednucleic acid comprising a nucleic acid fragment from position 1 toposition 3348 of SEQ ID NO:1 fused to a DNA sequence encoding a proteinof interest. Another embodiment of the invention is the vector comprisedof SEQ ID NO:1 encoding SEQ ID NO:2, the plant cell transformed with thevector, and the plant generated with the transformed plant cell.

[0033] In another aspect, the invention is a purified nucleic acidcomprising a nucleic acid fragment from position 1 to position 4831 ofSEQ ID NO:3 fused to a DNA sequence encoding a protein of interest,wherein expression of said purified nucleic acid in a plant cell resultsin a fusion protein having increased stability when compared to thestability of said protein of interest engineered without fusionexpressed in a plant cell of the same species. The invention is also theresulting fusion protein comprising SEQ ID NO:4 encoded by the purifiednucleic acid comprising a nucleic acid fragment from position 1 toposition 4831 of SEQ ID NO:3 fused to a DNA sequence encoding a proteinof interest. Another embodiment of the invention is the vector comprisedof SEQ ID NO:3 encoding SEQ ID NO:4, the plant cell transformed with thevector, and the plant generated with the transformed plant cell.

[0034] In another aspect, the invention is a purified nucleic acidcomprising a nucleic acid fragment from position 1 to position 3348 ofSEQ ID NO:5 fused to a DNA sequence encoding a protein of interest,wherein expression of said purified nucleic acid in a plant cell resultsin a fusion protein having increased or decreased stability whencompared to the stability of said protein of interest engineered withoutfusion expressed in a plant cell of the same species. The purifiednucleic acid comprising a nucleic acid fragment from position 1 toposition 3348 of SEQ ID NO:5 fused to a DNA sequence encoding a proteinof interest could also have increased or decreased stability whencompared to the stability of the protein of interest fused to a nucleicacid fragment from position 1 to position 3348 of SEQ ID NO:1 expressedin a plant cell of the same species. Nucleotides at positions 1096-1098of SEQ ID NO:5 encode alanine or tyrosine. The invention is also theresulting fusion protein comprising SEQ ID NO:6 encoded by the purifiednucleic acid comprising a nucleic acid fragment from position 1 toposition 3348 of SEQ ID NO:5 fused to a DNA sequence encoding a proteinof interest. Another embodiment of the invention is the vector comprisedof SEQ ID NO:5 encoding SEQ ID NO:6, the plant cell transformed with thevector, and the plant generated with the transformed plant cell.

[0035] In another aspect, the invention is also a purified nucleic acidcomprising a nucleic acid fragment from position 1 to position 4831 ofSEQ ID NO:7 fused to a DNA sequence encoding a protein of interest,wherein expression of said purified nucleic acid in a plant cell resultsin a fusion protein having increased or decreased stability whencompared to the stability of said protein of interest engineered withoutfusion expressed in a plant cell of the same species. The purifiednucleic acid comprising a nucleic acid fragment from position 1 toposition 4831 of SEQ ID NO:7 fused to a DNA sequence encoding a proteinof interest could also have increased or decreased stability whencompared to the stability of the protein of interest fused to a nucleicacid fragment from position 1 to position 3348 of SEQ ID NO:1 expressedin a plant cell of the same species. Nucleotides at positions 1096-1098of SEQ ID NO:5 encode alanine or tyrosine. The invention is also theresulting fusion protein comprising SEQ ID NO:8 encoded by the purifiednucleic acid comprising a nucleic acid fragment from position 1 toposition 4831 of SEQ ID NO:7 fused to a DNA sequence encoding a proteinof interest. Another embodiment of the invention is the vector comprisedof SEQ ID NO:7 encoding SEQ ID NO:8, the plant cell transformed with thevector, and the plant generated with the transformed plant cell.

[0036] In yet another aspect, the invention is a method for decreasingthe degradation rate of an engineered protein of interest in a plantcell comprising the steps a) constructing a vector comprising a nucleicacid sequence that encodes a membrane binding protein from theSindbis-like plant virus family fused to a nucleotide sequence encodingthe protein of interest, the vector expressible in a plant cell; and b)introducing and expressing he vector in the plant cell to form a fusedprotein, wherein the degradation rate of the fused protein is less thanthe degradation rate of the engineered protein of interest in the plantcell or a plant cell of the same species. The Sindbis-like plant virusfamily contains “WFP” motif as depicted at amino acid position 365-367of SEQ ID NO:2. The vector may be integrated into the genome of saidplant cell. The Sindbis-like plant virus is alfalfa mosaic virus, bromemosaic virus, citrus leaf rugose virus, cucumber mosaic virus, sunn-hempmosaic virus, tobacco mosaic virus, tobacco rattle virus, or turnip veinclearing virus. The invention further embodies a plant cell transformedaccording to this method and the plant generated from the transformedplant cell.

[0037] In another aspect, the invention also embodies a method forincreasing the degradation rate of an engineered protein of interest ina plant cell comprising the steps a) constructing a vector comprising anucleic acid sequence that encodes a membrane binding protein from theSindbis-like plant virus family fused to a nucleotide sequence encodingthe protein of interest, the vector expressible in a plant cell; and b)introducing and expressing he vector in the plant cell to form a fusedprotein, wherein the degradation rate of the fused protein is less thanthe degradation rate of the engineered protein of interest in the plantcell or a plant cell of the same species. The Sindbis-like plant virusfamily contains a mutation in the “WFP” motif as depicted at amino acidposition 365-367 of SEQ ID NO:2. The vector may be integrated into thegenome of said plant cell. The Sindbis-like plant virus is alfalfamosaic virus, brome mosaic virus, citrus leaf rugose virus, cucumbermosaic virus, sunn-hemp mosaic virus, tobacco mosaic virus, tobaccorattle virus, or turnip vein clearing virus. The invention furtherembodies a plant cell transformed according to this method and the plantgenerated from the transformed plant cell.

[0038] In another aspect, the invention is furthermore a purifiednucleic acid comprising a nucleic acid fragment encoding a membranebinding protein from the Sindbis-like plant virus fused to a DNAsequence encoding a protein of interest. The Sindbis-like plant virus isalfalfa mosaic virus, brome mosaic virus, citrus leaf rugose virus,cucumber mosaic virus, sunn-hemp mosaic virus, tobacco mosaic virus,tobacco rattle virus, and turnip vein clearing virus. The invention isthe resulting fusion protein encoded by a purified nucleic acidcomprising a nucleic acid fragment encoding a membrane binding proteinfrom the Sindbis-like plant virus fused to a DNA sequence encoding aprotein of interest. The resulting fusion protein comprising a membranebinding protein from the Sindbis-like plant virus family containing the“WFP” motif as depicted at amino acid position 365-367 of SEQ ID NO:2fused to an amino acid sequence of interest has increased stability overthe unfused protein of interest expressed in a cell of the same plantspecies. The invention is also the vector comprising a nucleic acidfragment encoding a membrane binding protein from the Sindbis-like plantvirus containing the “WFP” motif as depicted at amino acid position365-367 of SEQ ID NO:2 fused to a DNA sequence encoding a protein ofinterest. Additionally, the invention is the plant cell transformed withthe vector and the plant generated from the plant cell.

[0039] In another aspect, the invention is a purified nucleic acidcomprising a nucleic acid fragment encoding a membrane binding proteinfrom the Sindbis-like plant virus containing a mutation in the “WFP”motif as depicted at amino acid position 365-367 of SEQ ID NO:2 fused toa DNA sequence encoding a protein of interest. The Sindbis-like plantvirus is alfalfa mosaic virus, brome mosaic virus, citrus leaf rugosevirus, cucumber mosaic virus, sunn-hemp mosaic virus, tobacco mosaicvirus, tobacco rattle virus, and turnip vein clearing virus. Theinvention is also the resulting fusion protein encoded by a purifiednucleic acid comprising a nucleic acid fragment encoding a membranebinding protein from the Sindbis-like plant virus fused to a DNAsequence encoding a protein of interest. The resulting fusion proteincomprising a membrane binding protein from the Sindbis-like plant virusfamily containing a mutation in the “WFP” motif as depicted at aminoacid position 365-367 of SEQ ID NO:2 fused to an amino acid sequence ofinterest has increased or decreased stability over the unfused proteinof interest expressed in a cell of the same plant species. The inventionis also the vector comprising a nucleic acid fragment encoding amembrane binding protein from the Sindbis-like plant virus containing amutation in the “WFP” motif as depicted at amino acid position 365-367of SEQ ID NO:2 fused to a DNA sequence encoding a protein of interest.Additionally, the invention is the plant cell transformed with thevector and the plant generated from the plant cell.

DETAILED DESCRIPTION

[0040] We have identified an amino acid motif, “WFP”, from the TMV 126kDa and 183 kDa proteins (amino acid position 365 to 367 of SEQ ID:2 andSEQ ID NO:4) that is conserved among viral membrane—associated proteins.The TMV 126 kDa and 183 kDa proteins localize to the ER in infected N.tabacum and N. benthamiana cells. Mutating the “WFP” motif to “WYP” or“WAP” resulted in a variety of effects, somewhat dependent upon the hostspecies. Although the “WFP” motif causes a fused protein to resistubiquitin-mediated degradation, the mutant 126Y:GFP and 126A:GFPresulted in an increased degradation of a fused protein. Thus, wedisclose a method to modulate the rate, and therefore the stability, ofan engineered protein.

[0041] One method to decrease the rate of degradation of an engineeredprotein in plant cells includes creating a vector expressible in a plantcell, wherein the vector encodes a fusion protein between the TMV 126kDa protein and a protein of interest. An exemplary nucleotide sequencefor inclusion in this vector is SEQ ID NO:1 which encodes the TMV 126kDa protein of SEQ ID NO:2. The vector could be designed for transienttransfection, or for integration into the plant cell's genome. Aftercreating the vector expressible in a plant cell, the method includesintroducing the vector into one or more plant cells through anycurrently known methods of the art or other methods that will be known.The resulting plant cell containing the vector expresses the fusionprotein, which has a decreased rate of degradation compared to theprotein of interest when not expressed as a fusion protein. In additionto the method described for decreasing the rate of degradation of aprotein of interest, the invention as disclosed herein also includes thevector created for implementing the disclosed method, the nucleotidesequence that encodes the fusion protein, the fusion protein thatresults from the expression of the created vector, the plant cell orcells transformed with the created vector, and the plants that aregenerated from the transformed cells.

[0042] Another method to decrease the rate of degradation of anengineered protein in plant cells includes creating a vector expressiblein a plant cell, wherein the vector encodes a fusion protein between theTMV 183 kDa protein and a protein of interest. An exemplary nucleotidesequence for inclusion in this vector is SEQ ID NO:3 which encodes theTMV 186 kDa protein of SEQ ID NO:4. The vector could be designed fortransient transfection, or for integration into the plant cell's genome.After creating the vector expressible in a plant cell, the methodincludes introducing the vector into one or more plant cells through anycurrently known methods of the art or other methods that will be known.The resulting plant cell containing the vector expresses the fusionprotein, which has a decreased rate of degradation compared to theprotein of interest when not expressed as a fusion protein. In additionto the method described for decreasing the rate of degradation of aprotein of interest, the invention as disclosed herein also includes thevector created for implementing the disclosed method, the nucleotidesequence that encodes the fusion protein, the fusion protein thatresults from the expression of the created vector, the plant cell orcells transformed with the created vector, and the plants that aregenerated from the transformed cells.

[0043] The invention also includes methods to increase the rate ofdegradation of an engineered protein in plant cells. This methodincludes creating a vector expressible in a plant cell, wherein thevector encodes a fusion protein between a mutant TMV 126 kDa protein anda protein of interest. An exemplary nucleotide sequence for inclusion inthis vector is SEQ ID NO:5 which encodes a mutant TMV 126 kDa protein ofSEQ ID NO:6 where amino acid 366 is any amino acid but phenylalanine.Two exemplary amino acid substitutions include tyrosine and alanine. Thevector could be designed for transient transfection, or for integrationinto the plant cell's genome. After creating the vector expressible in aplant cell, the method includes introducing the vector into one or moreplant cells through any currently known methods of the art or othermethods that will be known. The resulting plant cell containing thevector expresses the fusion protein, which has an increased rate ofdegradation compared to the protein of interest when not expressed as afusion protein. In addition to the method described for increasing therate of degradation of a protein of interest, the invention as disclosedherein also includes the vector created for implementing the disclosedmethod, the nucleotide sequence that encodes the fusion protein, thefusion protein that results from the expression of the created vector,the plant cell or cells transformed with the created vector, and theplants that are generated from the transformed cells.

[0044] The invention also includes methods to increase the degradationrate of an engineered protein in plant cells. This method includescreating a vector expressible in a plant cell, wherein the vectorencodes a fusion protein between a mutant TMV 183 kDa protein and aprotein of interest. An exemplary nucleotide sequence for inclusion inthis vector is SEQ ID NO:7 which encodes a mutant TMV 183 kDa protein ofSEQ ID NO:8 where amino acid 366 is any amino acid but phenylalanine.Two exemplary amino acid substitutions include tyrosine and alanine. Thevector could be designed for transient transfection, or for integrationinto the plant cell's genome. After creating the vector expressible in aplant cell, the method includes introducing the vector into one or moreplant cells through any currently known methods of the art or othermethods that will be known. The resulting plant cell containing thevector expresses the fusion protein, which has an increased rate ofdegradation compared to the protein of interest when not expressed as afusion protein. In addition to the method described for increasing thedegradation rate of a protein of interest, the invention as disclosedherein also includes the vector created for implementing the disclosedmethod, the nucleotide sequence that encodes the fusion protein, thefusion protein that results from the expression of the created vector,the plant cell or cells transformed with the created vector, and theplants that are generated from the transformed cells.

[0045] As anyone skilled in the art can recognize, other nucleotidesequences that encode amino acid sequences with analogous function andhomologous sequence to TMV's 126/183 kDa protein may be used to decreasethe degradation rate of an engineered protein. This method to decreasethe degradation rate of an engineered protein in plant cells includescreating a vector expressible in a plant cell, wherein the vectorencodes a fusion protein between a protein with analogous function andhomologous sequence to TMV's 126/183 kDa protein from one of thefollowing Sindbis-like plant viruses: alfalfa mosaic virus, brome mosaicvirus, citrus leaf rugose virus, cucumber mosaic virus, sunn-hemp mosaicvirus, tobacco mosaic virus, tobacco rattle virus, and turnip veinclearing virus. The vector could be designed for transient transfection,or for integration into the plant cell's genome. After creating thevector expressible in a plant cell, the method includes introducing thevector into one or more plant cells through any currently known methodsof the art or other methods that will be known. The resulting plant cellcontaining the vector expresses the fusion protein, which has adecreased degradation rate compared to the protein of interest when notexpressed as a fusion protein. In addition to the method described fordecreasing the degradation rate of a protein of interest, the inventionas disclosed herein also includes the vector created for implementingthe disclosed method, the nucleotide sequence that encodes the fusionprotein, the fusion protein that results from the expression of thecreated vector, the plant cell or cells transformed with the createdvector, and the plants that are generated from the transformed cells.

[0046] Yet another method to increase the degradation rate of anengineered protein in plant cells includes creating a vector expressiblein a plant cell, wherein the vector encodes a fusion protein between amutated protein with analogous function and homologous sequence to TMV's126/183 kDa protein from one of the following Sindbis-like plantviruses: alfalfa mosaic virus, brome mosaic virus, citrus leaf rugosevirus, cucumber mosaic virus, sunn-hemp mosaic virus, tobacco mosaicvirus, tobacco rattle virus, and turnip vein clearing virus. The vectorcould be designed for transient transfection, or for integration intothe plant cell's genome. After creating the vector expressible in aplant cell, the method includes introducing the vector into one or moreplant cells through any currently known methods of the art or othermethods that will be known. The resulting plant cell containing thevector expresses the fusion protein, which has an increased degradationrate compared to the protein of interest when not expressed as a fusionprotein. In addition to the method described for increasing thedegradation rate of a protein of interest, the invention as disclosedherein also includes the vector created for implementing the disclosedmethod, the nucleotide sequence that encodes the fusion protein, thefusion protein that results from the expression of the created vector,the plant cell or cells transformed with the created vector, and theplants that are generated from the transformed cells.

[0047] Materials and Methods

[0048] Plant Materials

[0049]Nicotiana benthamiana and Nicotiana tabacum Xanthi “nn” and “NN”were germinated in a tray and individually transplanted into 12 cm potscontaining an artificial soil medium (Metro-Mix 350, Grace). Plants weregrown in the greenhouse until needed under the following conditions: 16hour and 25° C. days and 8 hour and 17° C. nights. Supplemental lightintensity was 500 μmol photons M⁻² s⁻¹. Plants used for inoculationexperiments were six to seven weeks old. Although other conditions maybe used, the above growth conditions are preferred.

[0050] Suspension Cells, Protoplasts and Transfection

[0051] The maintenance of suspension cells, preparation of protoplastsand transfection of protoplasts by electroporation were conductedaccording to Watanabe et al.(1987), modified for electroporating the N.benthamiana cells and protoplasts. N. tabacum BY-2 (Dr. Richard Cyr,Penn State University) suspension cells were grown in 50 ml of culturemedia (4.3 g/L M&S salt, 100 mg/L myo-inositol, 1 mg/L thiamine, 0.2mg/L 2,4-D, 255 mg/L KH₂PO₄, 30 g/L sucrose, pH 5.0) at 26° C.constantly shaking at 150 rpm and sub-cultured weekly. Suspension cellsof N. benthamiana (Dr. Bryce Falk, University

[0052] California—Davis) were grown in culture media (4.3 g/L M&S salt,0.204 g/L KH₂PO₄, 100 mg/L myo-inositol, 0.2 mg/L 2,4-D, 0.1 mg/LKinetin, 1 mg/L thiamine, 0.5 mg/L pyridoxide, 0.5 mg/L nicotinic acid,30 g/L sucrose, pH 5.8) at 26° C. constantly shaking at 150 rpm andsub-cultured every 10 days.

[0053] In order to create protoplasts, both BY-2 and N. benthamianacells were digested with 1% Cellulose, R-10; 0.1% Pectolyase Y-23 and 1%Driselase (Karlan) in MMC buffer (13% mannitol, 5 mM MES, 10 mM CaCl₂,pH 5.8) at room temperature for 3 hours. The digested cells wereoverlaid on a 20.5% sucrose cushion and spun at 1,100 rpm on an IECcentrifuge for 11 minutes. The protoplasts on top of the cushion werecollected and washed twice with MMC buffer. About 1×10⁶ protoplasts wereresuspended in 0.8 ml of the electroporation buffer (13% mannitol, 70 mMKCl, 5 mM MES, pH 5.8).

[0054] Fifteen μg of plasmid DNA or 5 μg of in vitro transcript viralRNA (see below) were mixed with 0.8 ml of protoplasts in a precooledcuvette and electroporated with the following setting: 250V, 220 μF and50 mS (ProGenetor II, Hoefer Scientific Instruments, San Francisco,Calif. USA ). After electroporation, protoplasts were incubated on icefor 10 minutes and washed with 2 ml of MMC buffer. The transfectedprotoplasts were resuspended in 3 ml of culture media with 13% mannitoland incubated at 26° C. in the dark for BY-2 protoplasts or under lightfor N. benthamiana protoplasts. Although alternative methods may beemployed, the above methods for maintaining suspension cells, creatingprotoplasts, and transfecting both are preferred.

[0055] In Vitro Site-Directed Mutagenesis

[0056] To mutate the second amino acid in the “WFP” motif, in vitrosite-directed mutagenesis was performed as described before (Bao et al,1996). The phenylalanine in the “WFP” motif from M^(IC)m2 (an infectioustranscript of M^(IC) TMV altered at a single nucleotide to the UI strainsequence in the 126 kDa protein open reading frame) (Shintaku et al.,1996) was replaced with alanine and tyrosine, respectively. In order tocreate the “WAP” motif, in vitro site-directed mutagenesis was performedusing the following primer complementary to nucleotides 1141-1177:5′-CTCATTTCGGGAGCCCAGTAATTGACTGATGATGAAT-3′ (SEQ ID NO:22). In order tocreate the “WYP” motif, in vitro site-directed mutagenesis was performedusing the following primer complementary to nucleotides 1141-1173:5′-TTTCGGGATACCAGTAATTGACTGATGATGAAT-3′ (SEQ ID NO:23). The underlinedcodon indicates the mutated sites. All mutant clones were confirmed tocontain the specified alteration by sequence analysis. Althoughsite-directed mutagenesis to the WAP and WYP motifs may be performedusing alternative primers, the above methods are preferred.Additionally, other mutations can be made in the place of phenylalanine366 as numbered in SEQ ID NO:2 in the same way.

[0057] In Vitro Transcription and Inoculation

[0058] Plasmid DNA of infectious TMV cDNA clones was linearized by Acc65I and gel-purified to act as a template in the in vitro transcriptionreaction performed as described previously (Shintaku et al., 1996). 5 μgof transcript viral RNA was inoculated on the mature leaves of N.benthamiana, N. tabacum Xanthi “NN”, and “nn” which were dusted with theabrasive carborundum. The inoculated plants were kept in the greenhouseto observe local lesions and systemic symptoms. Other method may beutilized for in vitro transcription and inoculation, but the processesdescribed above are preferred.

[0059] Construction of 126 kDa-GFP Fusion Chimeric Vectors

[0060] A cDNA fragment encoding the 126 kDa protein of the M^(IC) TMVwas amplified from plasmid L19 (Shintaku et al., 1996) using the Pfupolymerase (Stratagene) and a pair of primers ST(5′-CCATGCCATGGCGCTCGAGATGGCATACACACAGACA-3′ (SEQ ID NO:24), where theunderlined nucleotides indicate the TMV genome sequence from theposition 69 to 86) and GT (5′-CCCTTGCTCACCATTTGTGTTCCTGCATCG-3′ (SEQ IDNO:25), where the underlined nucleotides indicate the sequencecomplementary to TMV genome sequence, from the position 3401 to 3416).Green fluorescent protein (GFP) (EGFP, Clontech Laboratories, Inc., PaloAlto, Calif.) was amplified from plasmid pEGFP (Clontech) using the Pfupolymerase and a pair of primers TG(5′-ATGCAGGAACACAAATGGTGAGCAAGGGCG-3′) (SEQ ID NO:26) and 3GFP(5′-CCATGCCATGGCTCGAGTTACTTGTACAGCTCGT-3′) (SEQ ID NO:27). The amplifiedfragments were gel-purified and mixed as the template for the fusion PCRusing the primers 5T and 3GFP (method described by Higuchi, 1990). ThePCR product was the fusion of the 126 kDa protein gene and the GFP genewhich was purified and digested with Nco I. The digested fragment waspurified and ligated with plasmid pRTL2 (Restrepo et al., 1990)previously digested with Nco I. The ligation mixture was transformedinto E. coli HB 101. The clone containing the insert having the correctorientation was identified by restriction digestion and sequencing, andnamed p126:GFP. To make the mutated 126K fusion protein construct, theinfectious cDNA clones of “WFP”, “WYP” and “WAP” were digested with MluI and Dra III, sequentially. The Mlu I-Dra III fragments from each ofthe clones were inserted into the same site of p126:GFP previouslydigested with Mlu I and Dra III. Those clones containing wild type “WFP”motif and the mutated motifs (“WYP” and “WAP”) were named p126F:GFP,p126Y:GFP and p126A:GFP, respectively. Although a variety of methodscould be utilized to create chimeric vectors, the above methods arepreferred. Although only the full length 126 kDa protein was fused to agene of interest, this application anticipates that truncated portionsof the TMV 126 kDa protein or peptides can also be employed in thepresent invention as long as the amino acid sequence that stabilizes thefusion protein contains the “WFP” motif or elements that act in the samefashion.

[0061] Biolistic Bombardment and Fluorescent Microscopy

[0062] Transient expression of 126 kDa-GFP fusion protein in tobaccoleaves by biolistic bombardment was performed according to Itaya, et al.(1997). Five μg of each of p126F:GFP, p126Y:GFP and p126A:GFP wasbombarded into the lower epidermis of N. benthamiana and N. tabacumXanthi nn leaves using a Biolistic PDS 1000/He System (Bio-Rad) at apressure of 1,100 psi. The bombarded leaves were incubated in a sealedpetri dish with several pieces of water-soaked filter paper at 25° C.with light overnight.

[0063] The leaves were observed under a Nikon Microphot-FXepifluorescent microscope with a filter set B-2A, consisting of a blueexcitation filter (450-490 nm), a dichroic mirror (510 nm) and a barrierfilter (520 nm). Fluorescent images were photographed with the camerasystem attached to the microscope using Kodak Royal 400 color film.While biolistic bombardment and fluorescent microscopy could beaccomplished in different ways, the above methods are preferred.

[0064] Transient Expression of 126F:GFP, 126Y:GFP and 126A:GFP inProtoplasts

[0065] Fifteen μg of plasmid DNA of the three fusion protein constructs(126F:GFP, 126Y:GFP and 126A:GFP) were transfected into protoplasts ofN. benthamiana and BY-2 cells by electroporation as described above. Thetransfected protoplasts were collected at 7, 12, 16, 18, 24, 36, 48, 72,and 96 hours post-incubation and plated on a 12-well slide for a singlecell time course observation with a procedure as described previously(Mas and Beachy, 1998). The fluorescent fusion protein expression in theprotoplasts was examined by confocal laser scanning microscopy (CLSM) asdescribed below.

[0066] Immunofluorescent Labeling

[0067] Immunofluorescent labeling of TMV 126K protein and hostcomponents was conducted according to Heinlein et al. (1995) with aminor modification as follows. First, 0.5 ml of protoplasts of N.benthamiana and BY-2 infected with “WFP”, “WYP” and “WAP” viruses wereharvested 2 days post-infection. The protoplasts were spun down at 700rpm in 14 ml tubes (Falcon) at room temperature for 2 minutes andresuspended in fixative buffer (50 mM Na₂HPO₄, pH 6.7; 4%paraformadehyde, 0.1% glutaradehyde, 5 mM EGTA, pH 8.0) for 30 minutesat room temperature. The fixed protoplasts were plated on the slidesprecoated with 0.1% poly-L-lysine and then extracted with cold methanolfor 10 minutes. All washes were performed in phosphate-buffered saline(PBS), pH 7.0, containing 0.5% Tween-20 and 5 mM EGTA. Primaryantibodies were polyclonal rabbit IgG recognizing the TMV 126K protein(Nelson, et al. 1993) and polyclonal rabbit IgG against BiP, an ERassociated protein indicator, kindly provided by Dr. Becky Boston, NorthCarolina State University. Secondary antibodies were FITC-conjugatedgoat anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG(Molecular Probes, Eugene, Oreg., USA). The samples were mounted withmounting media ( 0.1 M Tris-HCl, pH 9.0; 50% glycerol, 1 mg/mlp-phenylenediamine) and stored at 4° C. before observation. Othermethods and materials may be used to visualize fusion protein presenceand localization, but the above methods and materials are preferred.

[0068] Proteosome Inhibition

[0069] ALLN (N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal, SigmaChemical Co. St. Louis, Mo.) was used at a final concentration of 75 μMin dimethyl sulfoxide (DMSO). The BY-2 protoplasts transfected withfusion protein constructs were incubated in the culture media containing75 μM of ALLN and collected 12, 24, and 48 hours post-transfection. Thetransient fluorescent protein expression in protoplasts was examined byCLSM as described below. There may be other ways to perform theinhibitor experiment, but the above methods are merely preferred.

[0070] Confocal Microscopy

[0071] Immnunofluorescent labeling signals and transient expression of126 kDa:GFP fusion protein in protoplasts were examined with CLSM (Chenget al., 2000). Most images were captured with 3% laser power, but in theinhibitor experiment, 10% laser power was used. The above conditions aremerely representative of conditions used to visualize data with confocalmicroscopy.

EXAMPLE 1

[0072] To better understand how the domains within the TMV 126 kDaprotein influence pathophysiology, the sequence of the TMV 126 kDaprotein was compared to functionally related proteins from otherSindbis-like plant viruses: alfalfa mosaic virus, brome mosaic virus,citrus leaf rugose virus, cucumber mosaic virus, sunn-hemp mosaic virus,tobacco rattle virus, and turnip vein clearing virus. The TMV 126 kDaprotein was aligned with its functional analogues from otherSindbis-like plant viruses using the CLUSTAL W program (Thompson et al.,1994) to identify a conserved “WFP” sequence(trypotophan-phenylalanine-proline) (FIG. 1). The “WFP” sequence iscontained within Domain I, between the methyltransferase and helicasedomains of this protein (FIG. 2A). This “WFP” sequence was also found inseveral plant proteins, most of which are membrane-associated. A personskilled in the art, understanding concepts of amino acid homology andfunctionally analogous proteins, will also recognize that the alignmentof FIG. 1 identifies parts of other sequences that may be fused tostabilize an engineered protein. Like the TMV 126 kDa protein usedherein, some of the proteins in FIG. 1 have a putative ER-colocalizingsignal that may be mutated to destabilize a fused engineered protein.

[0073] To create a destabilizing motif, three mutant viruses wereconstructed that were altered within this motif (FIG. 2B). The WFP virusrefers to a virus with a masked (M^(IC)) genetic background, except fora “Ser” residue, found in the U1 strain, at position 325 (Shintaku etal., 1996). This sequence alteration results in the WFP virus (alsoreferred to as M^(IC) m²) inducing severe symptoms and accumulating moreefficiently in systemic tissue than the parental M^(IC) virus (Derricket al., 1997). The WAP and WYP viruses were constructed by replacing“Phe” with “Ala” or “Tyr”, respectively, of the 126 kDa protein (FIG.2B). Both mutations of the “WFP” motif resulted in a virus unable tocause symptoms of the parental Tobacco mosaic virus. Although onlyalanine and tyrosine were substituted for phenylalanine in this presentexample, any substitute amino acid not having phenylalaninecharacteristics is anticipated in this invention because it acts todestabilize the fused protein.

[0074] Changing the phenylalanine to either alanine or tyrosine in theWFP motif decreased the infectivity of the mutant viruses on tobaccospecies. The WAP virus did not infect N. tabacum plants, but did infectN. benthamiana plants (Table 1). The WYP virus induced only mildsystemic symptoms on N. tabacum plants but severe systemic symptoms onN. benthamiana. The wild-type WFP virus induced severe symptoms on bothNicotiana species (Table 1). On N. tabacum Xanthi “NN” plants, a locallesion host for TMV, the WYP virus induced tiny necrotic lesions at 24°C., whereas the WFP virus induced larger lesions (FIG. 3A). Hightemperature treatment of 32° C. for three days before returning to 24°C. blocked the necrotic response of Nicotiana, but did not affect thelesion size induced by the WYP virus on “NN” plants (FIG. 3B). The WFPvirus, however, induced larger lesions after returning to the lowertemperature (FIG. 3B). These data demonstrate that the “WFP” motifwithin the 126 kDa protein is required for efficient virus replicationand infection, and that the necrosis response does not limit theinfectivity of the WYP virus. TABLE 1 Summary of biological analyses ofthe WFP, WYP, and WAP viruses in Nicotiana tabacum and Nicotianabenthamiana Host Phenotypes WFP WAP WYP N. tabacum Replication + − + N.tabacum Cell to cell + − + movement N. tabacum Systemic severe nonemild^(a) symptoms N. benthamiana Replication + − + N. benthamiana Cellto cell + − + movement N. benthamiana Systemic very severe mildsevere^(b) symptoms

[0075] We immunolabeled N. tabacum (cv. BY-2) and infected with the WFP,WYP or WAP viruses using antibodies against the TMV 126 kDa and bindingprotein (BiP), an ER marker (FIGS. 4A-H). The TMV 126 kDa proteincontaining the “WFP” motif (both the WFP and M^(IC) viruses) localizedto subcellular bodies similar to those observed in cells probed withanti-BiP (FIGS. 4A, 4B, 4E, and 4F). Both the 126 kDa protein containingthe “WYP” motif (FIG. 4C) and BiP (FIG. 4D) failed to localize in N.tabacum cells inoculated with WYP virus. Interestingly, the TMV 126 kDaprotein was not detected at all in WAP virus-infected cells of N.tabacum. There was no TMV 126 kDa protein detected in the mock-infectedN. tabacum protoplast (FIG. 4G). In N. benthamiana protoplasts, the 126kDa proteins of the WYP and WAP viruses localized similarly to the 126kDa protein from the WFP virus (data not shown). These results indicatethat the “WFP” motif within the TMV 126 kDa protein is necessary for theproper interaction of the TMV 126 kDa protein with host factors tolocalize to the ER, and this association is correlated with the abilityof the virus to efficiently infect the host. Altering the “WFP” motifprevents localization to the ER.

EXAMPLE 2

[0076] The TMV 126 kDa protein ORFs from the “WFP”, “WYP”, and “WAP”viruses were fused with GFP ORF to yield 126F:GFP (containing the “WFP”motif), 126Y:GFP (containing the “WYP” motif) and 126A:GFP (containingthe “WAP” motif) constructs. These constructs were placed behind anenhanced 35S promoter for transient expression in both N. tabacum Xanthinn and N. benthamiana leaf cells by biolistic bombardment (FIG. 5A). Thefluorescent signal was observed in subcellular bodies as punctate dotsand along the periphery of the cells (FIGS. 5B-5S). The fluorescent126F:GFP was stable for at least 8 days in both Nicotiana species (FIGS.5B-5G), while the intensity of fluorescence declined rapidly for the126A:GFP and 126Y:GFP fusions in N. tabacum (FIG. 5H, 5J, 5L, 5N, 5P,and 5R). In N. benthamiana, however, the fluorescence produced by the126Y:GFP fusion was not reduced relative to the 126F:GFP fusion overtime (FIGS. 5S and 5G). The stability pattern of the various transientlyexpressed 126 kDa:GFP fusion proteins correlated with the ability of theparental and mutant viruses to efficiently infect the host. This findingalso shows that the stabilization of viral replicase complex through thealtered 126 kDa protein requires species-specific host factors.

[0077]N. benthamiana protoplasts were transfected with 126F:GFP-,126Y:GFP-, and 126A:GFP-containing plasmids to study the subcellularlocalization of the 126 kDa:GFP fusion proteins during transientexpression. The fusion proteins formed many small irregular bodieswithin the cytosol (FIGS. 6A, 6C, and 6E), unlike the non-fused GFPconstruct which failed to form subcellular bodies 7 hourspost-inoculation (FIG. 6G). At 24 hours after inoculation, theprotoplasts expressing the 126F:GFP and 126Y:GFP constructs appeared tohave fewer, but larger fluorescent bodies (FIGS. 6D and 6F). Theprotoplasts expressing free GFP formed no punctate bodies even after 24hours (FIGS. 6G and 6H).

[0078]N. tabacum (cv. BY-2) protoplasts were also transfected with126F:GFP-, 126Y:GFP-, and 126A:GFP-containing plasmids. The irregularfluorescent bodies that resulted could be categorized into two types:small bodies less than 2 μm in diameter which disappeared over time, andlarge bodies more than 2 μm in diameter which persisted. The wild-type126F:GFP fusion protein formed both types of bodies in BY-2 cells (FIGS.7A and 7B). The 126Y:GFP and 126A:GFP fusion proteins formed mostly onlysmall bodies (FIG. 7B). Generally, the 126A:GFP fusion protein producedfewer large bodies than did the 126Y:GFP fusion protein (FIG. 7A). Also,the small bodies produced by the 126A:GFP fusion protein disappearedeven more rapidly than did those formed by the 126Y:GFP fusion protein(FIG. 7A). These results indicated that the 126 kDa protein alone, evenwithout other viral proteins, localized to the ER in infected cells. Adeterminant that controls localization of TMV 126 kDa protein to the ERis the “WFP” motif or the motif affected by the “WFP” motif.

[0079] The previous results indicated that the altered 126 kDa:GFPfusion proteins were less stable than the “WFP” containing fusionprotein in BY-2 cells. To determine if the 26S proteosome wasresponsible for degrading these TMV proteins, we expressed the fusionproteins in BY-2 cells incubated in the presence or absence ofAcetyl-Leu-Leu-norleucinal (ALLN), an inhibitor of the 26S proteasome.Cells incubated in ALLN and transfected with either of the mutant126Y:GFP or 126A:GFP fusion constructs yielded fluorescent signals thatwere greater and more stable compared to the signals from transfectedcells without ALLN (compare FIGS. 8G, 8I, 8K, 8M, 8O, and 8Q to FIGS.8B, 8D, 8F, 8H, 8J, and 8L). In the ALLN-treated cells, the 126Y:GFPfusion protein produced more fluorescent small bodies and also formedthe large irregular bodies that localized around the nucleus at latestages, similar to what was observed for the 126F:GFP fusion protein(FIGS. 8G-8L for 126Y:GFP and compare to FIGS. 8B, 8D, and 8E for126F:GFP). This result demonstrates that the “WYP” fusion protein canform small bodies in the absence of ALLN, but cannot avoid the hostdegradation machinery in the absence of inhibitor, thereby leading to aninability to form the large stable bodies. Also, the presence of theinhibitor led to greater expression of the 126F: wild-type GFP fusionthan in its absence (FIG. 8B, 8D, 8F, versus 8A, 8C, and 8E). Thesefindings indicate that the instability of the altered 126 kDa:GFP fusionproteins was due to their degradation by the host 26S proteasome. Themaintenance of the “WFP” motif within the 126 kDa protein was thuscritical to inhibit the degradation of this protein by the hostubiquitin-facilitated pathway. The ability of the altered viral proteinsto form bodies in N. benthamiana cells and not in N. tabacum BY2 cellsshowed that the ability to degrade the viral protein is controlled byhost factors in N. tabacum that better recognize structural change inthe target than those from N. benthamiana. Therefore, protein with theWFP motif resists ubiquitin-dependent degradation.

[0080] We have found that the 126 kDa protein stabilizes expression of afused protein in cells. When the 126 kDa protein was fused with GFP, theexpression of the fused protein in the cell cytoplasm, as detected byfluorescence microscopy, was observed for two days longer than unfusedGFP. The free GFP was only detectable for up to 5 days, whereas the 126kDa protein fused with GFP was detectable at 7 days, the last time pointcollected. Thus, the fusion of the normal 126 kDa protein (i.e.containing the WFP motif) with a foreign protein stabilizes theexpression phenotype of the foreign protein.

[0081] In summary, an amino acid motif, “WFP”, was identified in the TMV126 kDa and 183 kDa proteins (amino acid position 365 to 367 as numberedSEQ ID:2 and SEQ ID NO:4) that was conserved among both viral proteinsand host membrane-associated proteins. When the “WFP” motif was mutatedto “WYP” or “WAP”, the mutant viruses containing these new motifs weredramatically less capable of infecting and replicating in N. tabacum,but could infect N. benthamiana. Immunolabeling of the 126 kDa/183 kDaprotein complex in virus-infected cells indicated that the replicaseco-localized with binding protein (BiP), a host protein associated withthe ER. However, the mutant virus containing WYP failed to localize BiPand the 126 kDa mutant protein to the ER. Transient expression of the126 kDa protein fused with GFP showed that the mutant 126Y:GFP and126A:GFP were unstable in plants and protoplasts of N. tabacum, butstable in plants and protoplasts of N. benthamiana. Thus, altering the“WFP” motif resulted in an increased degradation of this fusion proteindepending on the host cell species. The wild-type 126 kDa:GFP proteinfusions formed cytoplasmic bodies in transfected protoplasts and thesebodies could be categorized into two types. Small bodies were less than2 μm in diameter and disappeared in the WYP- and WAP-transfected cellsafter 48 hours, and large bodies that were more than 2 μm in diameterthat persisted for WFP-transfected cells but not for WYP - orWAP-transfected cells. The 126F:GFP fusion maintained expression oflarge bodies longer than did 126Y:GFP or 126A:GFP. In the presence ofthe 26S proteasome inhibitor (ALLN), the 126Y:GFP and 126A:GFP fusionsappeared more stable than in the absence of the inhibitor. Thus, theubiquitin degradation pathway is involved in the degradation of themutant 126 kDa protein. The accumulation of 126F:GFP fusion protein wasincreased in the presence of a 26S proteosome inhibitor, indicating someresistance of this protein, even in the absence of other viral proteins,to the ubiquitin degradation pathway.

EXAMPLE 3

[0082] Anyone skilled in the art of protein biochemistry recognizes thatthe invention herein disclosed may be combined with known methods andmaterials to yield embodiments not directly mentioned. Because a threeamino acid motif within a larger viral ER-colocalizing protein has beenidentified to render a fused protein more stable in plant cells, areasonable embodiment of the current invention is to alter the viralER-colocalizing protein in positions outside the three amino acid motif.By removing portions of the ER-colocalizing protein, it may be possibleto minimize the region that confers stability to a fused engineeredprotein. Alternatively, amino acid substitutions can be made at regionsoutside the three amino acid motif that confers stability to a fusedengineered protein. Naturally, because the truncations and substitutionsthat will be successful in the invention disclosed are outside the threeamino acid motif, they can be used with a mutated the three amino acidmotif to render a fused engineered protein unstable.

[0083] A person skilled in the art that recognizes the possibility ofincluding truncations and substitutions with the invention describedherein will also recognize the possibility of fusing a peptidecontaining within it the three amino acid motif to a gene of interest toconfer stability to the engineered protein. Alternatively, the samepeptide when identified may contain a mutated three amino acid motif torender a fused engineered protein unstable.

[0084] Literature Cited

[0085] Bao et al. 1996 J. Virol. 70: 6378-6383

[0086] Bao, Y. and Hull, R. 1993, J Gen Virol 74:1611-1616

[0087] Cheng et al., 2000, Plant J. 23: 1-16.

[0088] Cheng et al., 2000, Plant J., in press

[0089] Deom et al. Science, 1987, 237:389-394

[0090] Derrick et al., 1997, Mol. Plant-Microbe Interaction 10: 589-596.

[0091] Ecker et al. 1989 J Biol. Chem. 264:7715-779

[0092] Heinlein et al. 1995 Science 270: 1983-1985

[0093] Heinlein et al., 1998, Plant Cell 10: 1107-1120.

[0094] Holt, et al., 1990, MPMI 3:417-423

[0095] Higuchi, R. (1990) In “PCR Protocols: A guide to methods andapplications” (M. A. Innis, D. H. Gelford, J. J. Sninsky and T. J.White, Eds.) p. 177-183 Academic Press, San Diego

[0096] Itaya et al., 1997, Plant J. 12:1223-1230

[0097] Janda and Ahlquist 1998, Proc. Natl. Acad. Sci. USA 95: 2227-2232

[0098] Lewandowski and Dawson 2000, Virology 271: 90-98.

[0099] Laemmli 1970, Nature 227:680-685

[0100] Mas and Beachy 1998, Plant J 15:835-842

[0101] Mas and Beachy 1999, J. Cell Biol. 147: 945-958.

[0102] Nelson, et al. 1993 MPMI 6:45-54

[0103] Osman and Buck 1996, J. Virol. 70: 6227-7234.

[0104] Reichel and Beachy 2000, J. Virol. 74: 3330-3337.

[0105] Restrepo-Hartwig and Ahlquist 1999,J Virol. 73: 10303-10309.

[0106] Restrepo-Hartwig et al., 1990 Plant Cell 2:987-998

[0107] Shintaku et al., 1996, Virology 221: 218-225.

[0108] Sullivan and Ahlquist 1999, J. Virol. 73: 2622-2632

[0109] Szecsi et al., 1999, Mol. Plant-Microbe Interaction. 12: 143-152.

[0110] Thompson et al., 1994, Nucl. Acids Res. 22: 4673-4680.

[0111] Tpfer, et al. 1987, Nucl. Acids Res. 15:5890.

[0112] Vierstra, R. D. 1996 Plant Mol. Biol. 32:275-302

[0113] Watanabe et al. 1987 FEBS Letters 219:65-69

[0114] Watanabe et al., 1999, J. Virol. 73: 2633-2640.

1 27 1 3351 DNA Tobacco mosaic virus CDS (1)..(3348) 1 atg gca tac acacag aca gct acc aca tca gct ttg ctg gac act gtc 48 Met Ala Tyr Thr GlnThr Ala Thr Thr Ser Ala Leu Leu Asp Thr Val 1 5 10 15 cga gga aac aactcc ttg gtc aat gat cta gca aag cgt cgt ctt tac 96 Arg Gly Asn Asn SerLeu Val Asn Asp Leu Ala Lys Arg Arg Leu Tyr 20 25 30 gac aca gcg gtt gaagag ttt aac gct cgt gac cgc agg ccc aaa gtg 144 Asp Thr Ala Val Glu GluPhe Asn Ala Arg Asp Arg Arg Pro Lys Val 35 40 45 aac ttt tca aaa gta ataagc gag gag cag acg ctt att gct acc cgg 192 Asn Phe Ser Lys Val Ile SerGlu Glu Gln Thr Leu Ile Ala Thr Arg 50 55 60 gcg tat cca gaa ttc caa attaca ttt tat aac acg caa aat gcc gtg 240 Ala Tyr Pro Glu Phe Gln Ile ThrPhe Tyr Asn Thr Gln Asn Ala Val 65 70 75 80 cat tcg ctt gca ggt gga ttgcga tct tta gaa ctg gaa tat ctg atg 288 His Ser Leu Ala Gly Gly Leu ArgSer Leu Glu Leu Glu Tyr Leu Met 85 90 95 atg caa att ccc tac gga tca ttgact tat gac ata ggc ggg aat ttt 336 Met Gln Ile Pro Tyr Gly Ser Leu ThrTyr Asp Ile Gly Gly Asn Phe 100 105 110 gca tcg cat ctg ttc aag gga cgagca tat gta cac tgc tgc atg ccc 384 Ala Ser His Leu Phe Lys Gly Arg AlaTyr Val His Cys Cys Met Pro 115 120 125 aac ctg gac gtt cga gac atc atgcgg cat gaa ggc cag aaa gac agt 432 Asn Leu Asp Val Arg Asp Ile Met ArgHis Glu Gly Gln Lys Asp Ser 130 135 140 att gaa cta tac ctt tct agg ctagag aga ggg gga aaa aca gtc ccc 480 Ile Glu Leu Tyr Leu Ser Arg Leu GluArg Gly Gly Lys Thr Val Pro 145 150 155 160 aac ttc caa aag gaa gca tttgac aga tac gca gaa att cct gaa gac 528 Asn Phe Gln Lys Glu Ala Phe AspArg Tyr Ala Glu Ile Pro Glu Asp 165 170 175 gct gtc tgt cac aat act ttccag aca tgc gaa cat cag ccg atg caa 576 Ala Val Cys His Asn Thr Phe GlnThr Cys Glu His Gln Pro Met Gln 180 185 190 caa tca ggc aga gtg tat gccatt gcg cta cac agc ata tat gac ata 624 Gln Ser Gly Arg Val Tyr Ala IleAla Leu His Ser Ile Tyr Asp Ile 195 200 205 ccc gct gat gag ttc ggg gcagca ctc ttg agg aaa aat gtc cat acg 672 Pro Ala Asp Glu Phe Gly Ala AlaLeu Leu Arg Lys Asn Val His Thr 210 215 220 tgc tat gcc gct ttc cac ttctct gag aac ctg ctt ctt gaa gat tca 720 Cys Tyr Ala Ala Phe His Phe SerGlu Asn Leu Leu Leu Glu Asp Ser 225 230 235 240 tac gtc aat ctg gac gaaatc aac gcg tgt ttt tcg cgc gat gga gac 768 Tyr Val Asn Leu Asp Glu IleAsn Ala Cys Phe Ser Arg Asp Gly Asp 245 250 255 aag ttg acc ttt tct tttgca tca gag agt act ctt aat tac tgt cat 816 Lys Leu Thr Phe Ser Phe AlaSer Glu Ser Thr Leu Asn Tyr Cys His 260 265 270 agt tat tct aat att cttaag tat gtg tgc aaa act tac ttc ccg gcc 864 Ser Tyr Ser Asn Ile Leu LysTyr Val Cys Lys Thr Tyr Phe Pro Ala 275 280 285 tct aat aga gag gtt tacatg aag gag ttt tta gtc acc agg gtt aat 912 Ser Asn Arg Glu Val Tyr MetLys Glu Phe Leu Val Thr Arg Val Asn 290 295 300 acc tgg ttt tgt aag ttttct aga ata gat act ttt ctt ttg tac aaa 960 Thr Trp Phe Cys Lys Phe SerArg Ile Asp Thr Phe Leu Leu Tyr Lys 305 310 315 320 ggt gtg gcc cat aaaggt gta gat agt gag cag ttt tat act gca atg 1008 Gly Val Ala His Lys GlyVal Asp Ser Glu Gln Phe Tyr Thr Ala Met 325 330 335 gaa gac gca tgg cattac aaa aag act ctt gca atg tgc aac agc gag 1056 Glu Asp Ala Trp His TyrLys Lys Thr Leu Ala Met Cys Asn Ser Glu 340 345 350 aga atc ctc ctt gaggat tca tca aca gtc aat tac tgg ttt ccc gaa 1104 Arg Ile Leu Leu Glu AspSer Ser Thr Val Asn Tyr Trp Phe Pro Glu 355 360 365 atg agg gat atg gtcatc gta cca tta ttc gac att tct ttg gag act 1152 Met Arg Asp Met Val IleVal Pro Leu Phe Asp Ile Ser Leu Glu Thr 370 375 380 agt aag agg acg cgcaag gaa gtc tta gtg tcc aag gat ttc gtg ttt 1200 Ser Lys Arg Thr Arg LysGlu Val Leu Val Ser Lys Asp Phe Val Phe 385 390 395 400 aca gtg ctt aaccac att cga aca tac cag gca aaa gct ctt aca tac 1248 Thr Val Leu Asn HisIle Arg Thr Tyr Gln Ala Lys Ala Leu Thr Tyr 405 410 415 gta aat gtt ttgtcc ttc gtc gaa tcg att cga tcg agg gta atc att 1296 Val Asn Val Leu SerPhe Val Glu Ser Ile Arg Ser Arg Val Ile Ile 420 425 430 aac ggt gtg acagcg agg tcc gaa tgg gat gtg gac aaa tct ttg tta 1344 Asn Gly Val Thr AlaArg Ser Glu Trp Asp Val Asp Lys Ser Leu Leu 435 440 445 caa tcc ttg tccatg acg ttt tac ctg cat act aag ctt gcc gtt cta 1392 Gln Ser Leu Ser MetThr Phe Tyr Leu His Thr Lys Leu Ala Val Leu 450 455 460 aag gat gac ttactg att agc aag ttt agt ctc ggt tcg aaa acg gtg 1440 Lys Asp Asp Leu LeuIle Ser Lys Phe Ser Leu Gly Ser Lys Thr Val 465 470 475 480 tgc cag catgtg tgg gat gag att tca ctg gcg ttt ggg aac gca ttt 1488 Cys Gln His ValTrp Asp Glu Ile Ser Leu Ala Phe Gly Asn Ala Phe 485 490 495 ccc tcc gtgaaa gag agg ctc ttg aac agg aaa ctt atc aga gtg gca 1536 Pro Ser Val LysGlu Arg Leu Leu Asn Arg Lys Leu Ile Arg Val Ala 500 505 510 ggc gac gcacta gag atc agg gtg cct gat cta tat gtg acc ttc cac 1584 Gly Asp Ala LeuGlu Ile Arg Val Pro Asp Leu Tyr Val Thr Phe His 515 520 525 gac cga ttagtg act gag tac aag gcc tct gtg gac atg cct gcg ctt 1632 Asp Arg Leu ValThr Glu Tyr Lys Ala Ser Val Asp Met Pro Ala Leu 530 535 540 gac att aggaag aag atg gaa gaa acg gaa gtg atg tac aat gca ctt 1680 Asp Ile Arg LysLys Met Glu Glu Thr Glu Val Met Tyr Asn Ala Leu 545 550 555 560 tca gagtta tcg gtg tta agg gag tct gac aaa ttc gat gtt gat gtt 1728 Ser Glu LeuSer Val Leu Arg Glu Ser Asp Lys Phe Asp Val Asp Val 565 570 575 ttt tcccag atg tgc caa tct ttg gaa gtt gac gca atg acg gca gcg 1776 Phe Ser GlnMet Cys Gln Ser Leu Glu Val Asp Ala Met Thr Ala Ala 580 585 590 aag gttata gtc gcg gtc atg agc aat aag agc ggt ctg act ctc aca 1824 Lys Val IleVal Ala Val Met Ser Asn Lys Ser Gly Leu Thr Leu Thr 595 600 605 ttt gaacga cct act gag gcg aat gtt gcg cta gct tta cag gat caa 1872 Phe Glu ArgPro Thr Glu Ala Asn Val Ala Leu Ala Leu Gln Asp Gln 610 615 620 gaa aaggct tca gaa ggt gct ttg gta gtt acc tca aga gaa gtt gaa 1920 Glu Lys AlaSer Glu Gly Ala Leu Val Val Thr Ser Arg Glu Val Glu 625 630 635 640 gaaccg tcc atg aag ggt tcg atg gcc aga gga gag tta caa tta gct 1968 Glu ProSer Met Lys Gly Ser Met Ala Arg Gly Glu Leu Gln Leu Ala 645 650 655 ggtctt gct gga gat cat ccg gag tcg tcc tat tct agg aac gag gag 2016 Gly LeuAla Gly Asp His Pro Glu Ser Ser Tyr Ser Arg Asn Glu Glu 660 665 670 atagag tct tta gag cag ttt cat atg gca acg gca gat tcg tta att 2064 Ile GluSer Leu Glu Gln Phe His Met Ala Thr Ala Asp Ser Leu Ile 675 680 685 cgtaag cag atg agc tcg att gtg tac acg ggt ccg att aaa gtt cag 2112 Arg LysGln Met Ser Ser Ile Val Tyr Thr Gly Pro Ile Lys Val Gln 690 695 700 caaatg aaa aac ttt atc gat agc ctg gta gca tca cta tct gct gcg 2160 Gln MetLys Asn Phe Ile Asp Ser Leu Val Ala Ser Leu Ser Ala Ala 705 710 715 720gtg tcg aat ctc gtc aag atc ctc aaa gat aca gct gct att gac ctt 2208 ValSer Asn Leu Val Lys Ile Leu Lys Asp Thr Ala Ala Ile Asp Leu 725 730 735gaa acc cgt caa aag ttt gga gtc ttg gat gtt aca tct agg aag tgg 2256 GluThr Arg Gln Lys Phe Gly Val Leu Asp Val Thr Ser Arg Lys Trp 740 745 750tta att aaa cca acg gcc aag agt cat gca tgg ggt gtt gtt gaa acc 2304 LeuIle Lys Pro Thr Ala Lys Ser His Ala Trp Gly Val Val Glu Thr 755 760 765cac gcg agg aag tat cat gtg gcg ctt ctg gaa tat gat gag cag ggt 2352 HisAla Arg Lys Tyr His Val Ala Leu Leu Glu Tyr Asp Glu Gln Gly 770 775 780gtg gtg aca tgc gat gat tgg aga aga gta gct gtc agc tct gag tct 2400 ValVal Thr Cys Asp Asp Trp Arg Arg Val Ala Val Ser Ser Glu Ser 785 790 795800 gtt gtt tat tcc gac atg gcg aaa ctc aga act ctg cgc aga ctg ctt 2448Val Val Tyr Ser Asp Met Ala Lys Leu Arg Thr Leu Arg Arg Leu Leu 805 810815 cga aac gga gaa ccg cat gtc agt agc gca aag gtt gtt ctt gtg gac 2496Arg Asn Gly Glu Pro His Val Ser Ser Ala Lys Val Val Leu Val Asp 820 825830 gga gtt ccg ggc tgt gga aaa acc aaa gaa att ctt tcc agg gtt aat 2544Gly Val Pro Gly Cys Gly Lys Thr Lys Glu Ile Leu Ser Arg Val Asn 835 840845 ttt gat gaa gat cta att tta gta cct ggg aag caa gct gct gaa atg 2592Phe Asp Glu Asp Leu Ile Leu Val Pro Gly Lys Gln Ala Ala Glu Met 850 855860 atc aga aga cgt gcg aat tcc tca ggg att att gtg gcc acg aag gac 2640Ile Arg Arg Arg Ala Asn Ser Ser Gly Ile Ile Val Ala Thr Lys Asp 865 870875 880 aac gtt aaa acc gtt gat tct ttc atg atg aat ttt ggg aaa agc aca2688 Asn Val Lys Thr Val Asp Ser Phe Met Met Asn Phe Gly Lys Ser Thr 885890 895 cgc tgt cag ttc aag agg tta ttc att gat gaa ggg ttg atg ttg cat2736 Arg Cys Gln Phe Lys Arg Leu Phe Ile Asp Glu Gly Leu Met Leu His 900905 910 act ggt tgt gtt aat ttt ctt gtg gcg atg tca ttg tgc gaa att gca2784 Thr Gly Cys Val Asn Phe Leu Val Ala Met Ser Leu Cys Glu Ile Ala 915920 925 tat gtt tac gga gac aca cag cag att cca tac atc aat aga gtt tca2832 Tyr Val Tyr Gly Asp Thr Gln Gln Ile Pro Tyr Ile Asn Arg Val Ser 930935 940 gga ttc ccg tac ccc gcc cat ttt gcc aaa ttg gaa gtt gac gag gtg2880 Gly Phe Pro Tyr Pro Ala His Phe Ala Lys Leu Glu Val Asp Glu Val 945950 955 960 gag aca cgc aga act act ctc cgt tgt cca gcc gat gtc aca cattat 2928 Glu Thr Arg Arg Thr Thr Leu Arg Cys Pro Ala Asp Val Thr His Tyr965 970 975 ctg aac agg aga tat gag ggc ttt gtc atg agc act tct tcg gttaaa 2976 Leu Asn Arg Arg Tyr Glu Gly Phe Val Met Ser Thr Ser Ser Val Lys980 985 990 aag tct gtt tcg cag gag atg gtc ggc gga gcc gcc gtg atc aatccg 3024 Lys Ser Val Ser Gln Glu Met Val Gly Gly Ala Ala Val Ile Asn Pro995 1000 1005 atc tca aaa ccc ttg cat ggc aag atc ctg act ttt acc caatcg 3069 Ile Ser Lys Pro Leu His Gly Lys Ile Leu Thr Phe Thr Gln Ser1010 1015 1020 gat aaa gaa gct ctg ctt tca aga ggg tat tca gat gtt cacact 3114 Asp Lys Glu Ala Leu Leu Ser Arg Gly Tyr Ser Asp Val His Thr1025 1030 1035 gtg cat gaa gtg caa ggc gag aca tac tct gat gtt tca ctagtt 3159 Val His Glu Val Gln Gly Glu Thr Tyr Ser Asp Val Ser Leu Val1040 1045 1050 agg cta acc cct aca cca gtc tcc atc att gca gga gac agcccg 3204 Arg Leu Thr Pro Thr Pro Val Ser Ile Ile Ala Gly Asp Ser Pro1055 1060 1065 cat gtt ttg gtc gca ttg tca agg cac acc tgt tcg ctc aagtac 3249 His Val Leu Val Ala Leu Ser Arg His Thr Cys Ser Leu Lys Tyr1070 1075 1080 tac act gtt gtt atg gat cct tta gtt agt atc att aga gatcta 3294 Tyr Thr Val Val Met Asp Pro Leu Val Ser Ile Ile Arg Asp Leu1085 1090 1095 gag aaa ctt agc tcg tac ttg tta gat atg tat aag gtc gatgca 3339 Glu Lys Leu Ser Ser Tyr Leu Leu Asp Met Tyr Lys Val Asp Ala1100 1105 1110 gga aca caa tag 3351 Gly Thr Gln 1115 2 1116 PRT Tobaccomosaic virus 2 Met Ala Tyr Thr Gln Thr Ala Thr Thr Ser Ala Leu Leu AspThr Val 1 5 10 15 Arg Gly Asn Asn Ser Leu Val Asn Asp Leu Ala Lys ArgArg Leu Tyr 20 25 30 Asp Thr Ala Val Glu Glu Phe Asn Ala Arg Asp Arg ArgPro Lys Val 35 40 45 Asn Phe Ser Lys Val Ile Ser Glu Glu Gln Thr Leu IleAla Thr Arg 50 55 60 Ala Tyr Pro Glu Phe Gln Ile Thr Phe Tyr Asn Thr GlnAsn Ala Val 65 70 75 80 His Ser Leu Ala Gly Gly Leu Arg Ser Leu Glu LeuGlu Tyr Leu Met 85 90 95 Met Gln Ile Pro Tyr Gly Ser Leu Thr Tyr Asp IleGly Gly Asn Phe 100 105 110 Ala Ser His Leu Phe Lys Gly Arg Ala Tyr ValHis Cys Cys Met Pro 115 120 125 Asn Leu Asp Val Arg Asp Ile Met Arg HisGlu Gly Gln Lys Asp Ser 130 135 140 Ile Glu Leu Tyr Leu Ser Arg Leu GluArg Gly Gly Lys Thr Val Pro 145 150 155 160 Asn Phe Gln Lys Glu Ala PheAsp Arg Tyr Ala Glu Ile Pro Glu Asp 165 170 175 Ala Val Cys His Asn ThrPhe Gln Thr Cys Glu His Gln Pro Met Gln 180 185 190 Gln Ser Gly Arg ValTyr Ala Ile Ala Leu His Ser Ile Tyr Asp Ile 195 200 205 Pro Ala Asp GluPhe Gly Ala Ala Leu Leu Arg Lys Asn Val His Thr 210 215 220 Cys Tyr AlaAla Phe His Phe Ser Glu Asn Leu Leu Leu Glu Asp Ser 225 230 235 240 TyrVal Asn Leu Asp Glu Ile Asn Ala Cys Phe Ser Arg Asp Gly Asp 245 250 255Lys Leu Thr Phe Ser Phe Ala Ser Glu Ser Thr Leu Asn Tyr Cys His 260 265270 Ser Tyr Ser Asn Ile Leu Lys Tyr Val Cys Lys Thr Tyr Phe Pro Ala 275280 285 Ser Asn Arg Glu Val Tyr Met Lys Glu Phe Leu Val Thr Arg Val Asn290 295 300 Thr Trp Phe Cys Lys Phe Ser Arg Ile Asp Thr Phe Leu Leu TyrLys 305 310 315 320 Gly Val Ala His Lys Gly Val Asp Ser Glu Gln Phe TyrThr Ala Met 325 330 335 Glu Asp Ala Trp His Tyr Lys Lys Thr Leu Ala MetCys Asn Ser Glu 340 345 350 Arg Ile Leu Leu Glu Asp Ser Ser Thr Val AsnTyr Trp Phe Pro Glu 355 360 365 Met Arg Asp Met Val Ile Val Pro Leu PheAsp Ile Ser Leu Glu Thr 370 375 380 Ser Lys Arg Thr Arg Lys Glu Val LeuVal Ser Lys Asp Phe Val Phe 385 390 395 400 Thr Val Leu Asn His Ile ArgThr Tyr Gln Ala Lys Ala Leu Thr Tyr 405 410 415 Val Asn Val Leu Ser PheVal Glu Ser Ile Arg Ser Arg Val Ile Ile 420 425 430 Asn Gly Val Thr AlaArg Ser Glu Trp Asp Val Asp Lys Ser Leu Leu 435 440 445 Gln Ser Leu SerMet Thr Phe Tyr Leu His Thr Lys Leu Ala Val Leu 450 455 460 Lys Asp AspLeu Leu Ile Ser Lys Phe Ser Leu Gly Ser Lys Thr Val 465 470 475 480 CysGln His Val Trp Asp Glu Ile Ser Leu Ala Phe Gly Asn Ala Phe 485 490 495Pro Ser Val Lys Glu Arg Leu Leu Asn Arg Lys Leu Ile Arg Val Ala 500 505510 Gly Asp Ala Leu Glu Ile Arg Val Pro Asp Leu Tyr Val Thr Phe His 515520 525 Asp Arg Leu Val Thr Glu Tyr Lys Ala Ser Val Asp Met Pro Ala Leu530 535 540 Asp Ile Arg Lys Lys Met Glu Glu Thr Glu Val Met Tyr Asn AlaLeu 545 550 555 560 Ser Glu Leu Ser Val Leu Arg Glu Ser Asp Lys Phe AspVal Asp Val 565 570 575 Phe Ser Gln Met Cys Gln Ser Leu Glu Val Asp AlaMet Thr Ala Ala 580 585 590 Lys Val Ile Val Ala Val Met Ser Asn Lys SerGly Leu Thr Leu Thr 595 600 605 Phe Glu Arg Pro Thr Glu Ala Asn Val AlaLeu Ala Leu Gln Asp Gln 610 615 620 Glu Lys Ala Ser Glu Gly Ala Leu ValVal Thr Ser Arg Glu Val Glu 625 630 635 640 Glu Pro Ser Met Lys Gly SerMet Ala Arg Gly Glu Leu Gln Leu Ala 645 650 655 Gly Leu Ala Gly Asp HisPro Glu Ser Ser Tyr Ser Arg Asn Glu Glu 660 665 670 Ile Glu Ser Leu GluGln Phe His Met Ala Thr Ala Asp Ser Leu Ile 675 680 685 Arg Lys Gln MetSer Ser Ile Val Tyr Thr Gly Pro Ile Lys Val Gln 690 695 700 Gln Met LysAsn Phe Ile Asp Ser Leu Val Ala Ser Leu Ser Ala Ala 705 710 715 720 ValSer Asn Leu Val Lys Ile Leu Lys Asp Thr Ala Ala Ile Asp Leu 725 730 735Glu Thr Arg Gln Lys Phe Gly Val Leu Asp Val Thr Ser Arg Lys Trp 740 745750 Leu Ile Lys Pro Thr Ala Lys Ser His Ala Trp Gly Val Val Glu Thr 755760 765 His Ala Arg Lys Tyr His Val Ala Leu Leu Glu Tyr Asp Glu Gln Gly770 775 780 Val Val Thr Cys Asp Asp Trp Arg Arg Val Ala Val Ser Ser GluSer 785 790 795 800 Val Val Tyr Ser Asp Met Ala Lys Leu Arg Thr Leu ArgArg Leu Leu 805 810 815 Arg Asn Gly Glu Pro His Val Ser Ser Ala Lys ValVal Leu Val Asp 820 825 830 Gly Val Pro Gly Cys Gly Lys Thr Lys Glu IleLeu Ser Arg Val Asn 835 840 845 Phe Asp Glu Asp Leu Ile Leu Val Pro GlyLys Gln Ala Ala Glu Met 850 855 860 Ile Arg Arg Arg Ala Asn Ser Ser GlyIle Ile Val Ala Thr Lys Asp 865 870 875 880 Asn Val Lys Thr Val Asp SerPhe Met Met Asn Phe Gly Lys Ser Thr 885 890 895 Arg Cys Gln Phe Lys ArgLeu Phe Ile Asp Glu Gly Leu Met Leu His 900 905 910 Thr Gly Cys Val AsnPhe Leu Val Ala Met Ser Leu Cys Glu Ile Ala 915 920 925 Tyr Val Tyr GlyAsp Thr Gln Gln Ile Pro Tyr Ile Asn Arg Val Ser 930 935 940 Gly Phe ProTyr Pro Ala His Phe Ala Lys Leu Glu Val Asp Glu Val 945 950 955 960 GluThr Arg Arg Thr Thr Leu Arg Cys Pro Ala Asp Val Thr His Tyr 965 970 975Leu Asn Arg Arg Tyr Glu Gly Phe Val Met Ser Thr Ser Ser Val Lys 980 985990 Lys Ser Val Ser Gln Glu Met Val Gly Gly Ala Ala Val Ile Asn Pro 9951000 1005 Ile Ser Lys Pro Leu His Gly Lys Ile Leu Thr Phe Thr Gln Ser1010 1015 1020 Asp Lys Glu Ala Leu Leu Ser Arg Gly Tyr Ser Asp Val HisThr 1025 1030 1035 Val His Glu Val Gln Gly Glu Thr Tyr Ser Asp Val SerLeu Val 1040 1045 1050 Arg Leu Thr Pro Thr Pro Val Ser Ile Ile Ala GlyAsp Ser Pro 1055 1060 1065 His Val Leu Val Ala Leu Ser Arg His Thr CysSer Leu Lys Tyr 1070 1075 1080 Tyr Thr Val Val Met Asp Pro Leu Val SerIle Ile Arg Asp Leu 1085 1090 1095 Glu Lys Leu Ser Ser Tyr Leu Leu AspMet Tyr Lys Val Asp Ala 1100 1105 1110 Gly Thr Gln 1115 3 4834 DNATobacco mosaic virus gene (1)..(4831) 3 atggcataca cacagacagc taccacatcagctttgctgg acactgtccg aggaaacaac 60 tccttggtca atgatctagc aaagcgtcgtctttacgaca cagcggttga agagtttaac 120 gctcgtgacc gcaggcccaa agtgaacttttcaaaagtaa taagcgagga gcagacgctt 180 attgctaccc gggcgtatcc agaattccaaattacatttt ataacacgca aaatgccgtg 240 cattcgcttg caggtggatt gcgatctttagaactggaat atctgatgat gcaaattccc 300 tacggatcat tgacttatga cataggcgggaattttgcat cgcatctgtt caagggacga 360 gcatatgtac actgctgcat gcccaacctggacgttcgag acatcatgcg gcatgaaggc 420 cagaaagaca gtattgaact atacctttctaggctagaga gagggggaaa aacagtcccc 480 aacttccaaa aggaagcatt tgacagatacgcagaaattc ctgaagacgc tgtctgtcac 540 aatactttcc agacatgcga acatcagccgatgcaacaat caggcagagt gtatgccatt 600 gcgctacaca gcatatatga catacccgctgatgagttcg gggcagcact cttgaggaaa 660 aatgtccata cgtgctatgc cgctttccacttctctgaga acctgcttct tgaagattca 720 tacgtcaatc tggacgaaat caacgcgtgtttttcgcgcg atggagacaa gttgaccttt 780 tcttttgcat cagagagtac tcttaattactgtcatagtt attctaatat tcttaagtat 840 gtgtgcaaaa cttacttccc ggcctctaatagagaggttt acatgaagga gtttttagtc 900 accagggtta atacctggtt ttgtaagttttctagaatag atacttttct tttgtacaaa 960 ggtgtggccc ataaaggtgt agatagtgagcagttttata ctgcaatgga agacgcatgg 1020 cattacaaaa agactcttgc aatgtgcaacagcgagagaa tcctccttga ggattcatca 1080 acagtcaatt actggtttcc cgaaatgagggatatggtca tcgtaccatt attcgacatt 1140 tctttggaga ctagtaagag gacgcgcaaggaagtcttag tgtccaagga tttcgtgttt 1200 acagtgctta accacattcg aacataccaggcaaaagctc ttacatacgt aaatgttttg 1260 tccttcgtcg aatcgattcg atcgagggtaatcattaacg gtgtgacagc gaggtccgaa 1320 tgggatgtgg acaaatcttt gttacaatccttgtccatga cgttttacct gcatactaag 1380 cttgccgttc taaaggatga cttactgattagcaagttta gtctcggttc gaaaacggtg 1440 tgccagcatg tgtgggatga gatttcactggcgtttggga acgcatttcc ctccgtgaaa 1500 gagaggctct tgaacaggaa acttatcagagtggcaggcg acgcactaga gatcagggtg 1560 cctgatctat atgtgacctt ccacgaccgattagtgactg agtacaaggc ctctgtggac 1620 atgcctgcgc ttgacattag gaagaagatggaagaaacgg aagtgatgta caatgcactt 1680 tcagagttat cggtgttaag ggagtctgacaaattcgatg ttgatgtttt ttcccagatg 1740 tgccaatctt tggaagttga cgcaatgacggcagcgaagg ttatagtcgc ggtcatgagc 1800 aataagagcg gtctgactct cacatttgaacgacctactg aggcgaatgt tgcgctagct 1860 ttacaggatc aagaaaaggc ttcagaaggtgctttggtag ttacctcaag agaagttgaa 1920 gaaccgtcca tgaagggttc gatggccagaggagagttac aattagctgg tcttgctgga 1980 gatcatccgg agtcgtccta ttctaggaacgaggagatag agtctttaga gcagtttcat 2040 atggcaacgg cagattcgtt aattcgtaagcagatgagct cgattgtgta cacgggtccg 2100 attaaagttc agcaaatgaa aaactttatcgatagcctgg tagcatcact atctgctgcg 2160 gtgtcgaatc tcgtcaagat cctcaaagatacagctgcta ttgaccttga aacccgtcaa 2220 aagtttggag tcttggatgt tacatctaggaagtggttaa ttaaaccaac ggccaagagt 2280 catgcatggg gtgttgttga aacccacgcgaggaagtatc atgtggcgct tctggaatat 2340 gatgagcagg gtgtggtgac atgcgatgattggagaagag tagctgtcag ctctgagtct 2400 gttgtttatt ccgacatggc gaaactcagaactctgcgca gactgcttcg aaacggagaa 2460 ccgcatgtca gtagcgcaaa ggttgttcttgtggacggag ttccgggctg tggaaaaacc 2520 aaagaaattc tttccagggt taattttgatgaagatctaa ttttagtacc tgggaagcaa 2580 gctgctgaaa tgatcagaag acgtgcgaattcctcaggga ttattgtggc cacgaaggac 2640 aacgttaaaa ccgttgattc tttcatgatgaattttggga aaagcacacg ctgtcagttc 2700 aagaggttat tcattgatga agggttgatgttgcatactg gttgtgttaa ttttcttgtg 2760 gcgatgtcat tgtgcgaaat tgcatatgtttacggagaca cacagcagat tccatacatc 2820 aatagagttt caggattccc gtaccccgcccattttgcca aattggaagt tgacgaggtg 2880 gagacacgca gaactactct ccgttgtccagccgatgtca cacattatct gaacaggaga 2940 tatgagggct ttgtcatgag cacttcttcggttaaaaagt ctgtttcgca ggagatggtc 3000 ggcggagccg ccgtgatcaa tccgatctcaaaacccttgc atggcaagat cctgactttt 3060 acccaatcgg ataaagaagc tctgctttcaagagggtatt cagatgttca cactgtgcat 3120 gaagtgcaag gcgagacata ctctgatgtttcactagtta ggctaacccc tacaccagtc 3180 tccatcattg caggagacag cccgcatgttttggtcgcat tgtcaaggca cacctgttcg 3240 ctcaagtact acactgttgt tatggatcctttagttagta tcattagaga tctagagaaa 3300 cttagctcgt acttgttaga tatgtataaggtcgatgcag gaacacaata gcaattacag 3360 attgactcgg tgttcaaagg ttccaatctttttgtggcag cgccaaagac tggtgatatt 3420 tctgatatgc agttttacta tgataagtgtctcccaggca acagcaccat gatgaataat 3480 tttgatgctg ttaccatgag gttgactgacatttcattga atgtcaaaga ttgcatattg 3540 gatatgtcta agtctgttgc tgcgcctaaggatcaaatca aaccactaat acctatggta 3600 cgaacggcgg cagaaatgcc acgccagactggactattgg aaaatttagt ggcgatgatt 3660 aaaaggaact ttaacgcacc cgagttgtctggcatcattg atattgaaaa tactgcatct 3720 ttagttgtag ataagttttt cgatagttatttgcttaaag aaaaaagaaa accaaataaa 3780 aatgtttctt tgttcagtag agagtctctcaatagatggt tagaaaagca ggaacaggta 3840 acaataggcc agctcgcaga ttttgattttgtagatttgc cagcagttga tcagtacaga 3900 cacatgatca aagcacaacc caagcaaaaattggacactt caatccaaac ggagtacccg 3960 gctttgcaga cgattgtgta ccattcgaaaaagatcaatg caatatttgg cccgttgttt 4020 agtgagctta ctaggcaatt actggacagtgttgattcga gcagattttt gtttttcaca 4080 agaaagacac cagcgcagat tgaggatttcttcggagatc tcgacagtca tgtgccgatg 4140 gatgtcttgg agctggatat atcaaaatacgacaaatctc agaatgaatt ccactgtgca 4200 gtagaatacg agatttggcg aagattgggttttgaagact tcttgggaga agtttggaaa 4260 caagggcata gaaagaccac cctcaaggattataccgcag gtatcaaaac ttgcatctgg 4320 tatcaaagaa agagtgggga cgtcacgacattcattggaa acactgtgat cattgctgca 4380 tgtttggcct cgatgcttcc gatggagaaaataatcaaag gagccttttg tggtgacgat 4440 agtctgctgt acttcccaaa gggttgtgagtttccggatg tgcaacactc cgcgaatctt 4500 atgtggaatt ttgaagcaaa actgtttaaaaaacagtatg gatacttttg cggaagatat 4560 gtaatacatc acgacagagg atgcattgtgtattacgatc ccctaaagtt gatctcgaaa 4620 cttggcgcta aacacatcaa ggattgggaacacttggagg agttcagaag gtctctttgt 4680 gatgttgctg tttcgttgaa caattgtgcgtattatacac agttggacga cgctgtatgg 4740 gaggttcata agaccgcccc tccaggttcgtttgtttata aaagtctggt gaagtatttg 4800 tctgataaag ttctttttag aagtttgtttatag 4834 4 1616 PRT Tobacco mosaic virus misc_feature (1117)..(1117)Xaa is unknown 4 Met Ala Tyr Thr Gln Thr Ala Thr Thr Ser Ala Leu Leu AspThr Val 1 5 10 15 Arg Gly Asn Asn Ser Leu Val Asn Asp Leu Ala Lys ArgArg Leu Tyr 20 25 30 Asp Thr Ala Val Glu Glu Phe Asn Ala Arg Asp Arg ArgPro Lys Val 35 40 45 Asn Phe Ser Lys Val Ile Ser Glu Glu Gln Thr Leu IleAla Thr Arg 50 55 60 Ala Tyr Pro Glu Phe Gln Ile Thr Phe Tyr Asn Thr GlnAsn Ala Val 65 70 75 80 His Ser Leu Ala Gly Gly Leu Arg Ser Leu Glu LeuGlu Tyr Leu Met 85 90 95 Met Gln Ile Pro Tyr Gly Ser Leu Thr Tyr Asp IleGly Gly Asn Phe 100 105 110 Ala Ser His Leu Phe Lys Gly Arg Ala Tyr ValHis Cys Cys Met Pro 115 120 125 Asn Leu Asp Val Arg Asp Ile Met Arg HisGlu Gly Gln Lys Asp Ser 130 135 140 Ile Glu Leu Tyr Leu Ser Arg Leu GluArg Gly Gly Lys Thr Val Pro 145 150 155 160 Asn Phe Gln Lys Glu Ala PheAsp Arg Tyr Ala Glu Ile Pro Glu Asp 165 170 175 Ala Val Cys His Asn ThrPhe Gln Thr Cys Glu His Gln Pro Met Gln 180 185 190 Gln Ser Gly Arg ValTyr Ala Ile Ala Leu His Ser Ile Tyr Asp Ile 195 200 205 Pro Ala Asp GluPhe Gly Ala Ala Leu Leu Arg Lys Asn Val His Thr 210 215 220 Cys Tyr AlaAla Phe His Phe Ser Glu Asn Leu Leu Leu Glu Asp Ser 225 230 235 240 TyrVal Asn Leu Asp Glu Ile Asn Ala Cys Phe Ser Arg Asp Gly Asp 245 250 255Lys Leu Thr Phe Ser Phe Ala Ser Glu Ser Thr Leu Asn Tyr Cys His 260 265270 Ser Tyr Ser Asn Ile Leu Lys Tyr Val Cys Lys Thr Tyr Phe Pro Ala 275280 285 Ser Asn Arg Glu Val Tyr Met Lys Glu Phe Leu Val Thr Arg Val Asn290 295 300 Thr Trp Phe Cys Lys Phe Ser Arg Ile Asp Thr Phe Leu Leu TyrLys 305 310 315 320 Gly Val Ala His Lys Gly Val Asp Ser Glu Gln Phe TyrThr Ala Met 325 330 335 Glu Asp Ala Trp His Tyr Lys Lys Thr Leu Ala MetCys Asn Ser Glu 340 345 350 Arg Ile Leu Leu Glu Asp Ser Ser Thr Val AsnTyr Trp Phe Pro Glu 355 360 365 Met Arg Asp Met Val Ile Val Pro Leu PheAsp Ile Ser Leu Glu Thr 370 375 380 Ser Lys Arg Thr Arg Lys Glu Val LeuVal Ser Lys Asp Phe Val Phe 385 390 395 400 Thr Val Leu Asn His Ile ArgThr Tyr Gln Ala Lys Ala Leu Thr Tyr 405 410 415 Val Asn Val Leu Ser PheVal Glu Ser Ile Arg Ser Arg Val Ile Ile 420 425 430 Asn Gly Val Thr AlaArg Ser Glu Trp Asp Val Asp Lys Ser Leu Leu 435 440 445 Gln Ser Leu SerMet Thr Phe Tyr Leu His Thr Lys Leu Ala Val Leu 450 455 460 Lys Asp AspLeu Leu Ile Ser Lys Phe Ser Leu Gly Ser Lys Thr Val 465 470 475 480 CysGln His Val Trp Asp Glu Ile Ser Leu Ala Phe Gly Asn Ala Phe 485 490 495Pro Ser Val Lys Glu Arg Leu Leu Asn Arg Lys Leu Ile Arg Val Ala 500 505510 Gly Asp Ala Leu Glu Ile Arg Val Pro Asp Leu Tyr Val Thr Phe His 515520 525 Asp Arg Leu Val Thr Glu Tyr Lys Ala Ser Val Asp Met Pro Ala Leu530 535 540 Asp Ile Arg Lys Lys Met Glu Glu Thr Glu Val Met Tyr Asn AlaLeu 545 550 555 560 Ser Glu Leu Ser Val Leu Arg Glu Ser Asp Lys Phe AspVal Asp Val 565 570 575 Phe Ser Gln Met Cys Gln Ser Leu Glu Val Asp AlaMet Thr Ala Ala 580 585 590 Lys Val Ile Val Ala Val Met Ser Asn Lys SerGly Leu Thr Leu Thr 595 600 605 Phe Glu Arg Pro Thr Glu Ala Asn Val AlaLeu Ala Leu Gln Asp Gln 610 615 620 Glu Lys Ala Ser Glu Gly Ala Leu ValVal Thr Ser Arg Glu Val Glu 625 630 635 640 Glu Pro Ser Met Lys Gly SerMet Ala Arg Gly Glu Leu Gln Leu Ala 645 650 655 Gly Leu Ala Gly Asp HisPro Glu Ser Ser Tyr Ser Arg Asn Glu Glu 660 665 670 Ile Glu Ser Leu GluGln Phe His Met Ala Thr Ala Asp Ser Leu Ile 675 680 685 Arg Lys Gln MetSer Ser Ile Val Tyr Thr Gly Pro Ile Lys Val Gln 690 695 700 Gln Met LysAsn Phe Ile Asp Ser Leu Val Ala Ser Leu Ser Ala Ala 705 710 715 720 ValSer Asn Leu Val Lys Ile Leu Lys Asp Thr Ala Ala Ile Asp Leu 725 730 735Glu Thr Arg Gln Lys Phe Gly Val Leu Asp Val Thr Ser Arg Lys Trp 740 745750 Leu Ile Lys Pro Thr Ala Lys Ser His Ala Trp Gly Val Val Glu Thr 755760 765 His Ala Arg Lys Tyr His Val Ala Leu Leu Glu Tyr Asp Glu Gln Gly770 775 780 Val Val Thr Cys Asp Asp Trp Arg Arg Val Ala Val Ser Ser GluSer 785 790 795 800 Val Val Tyr Ser Asp Met Ala Lys Leu Arg Thr Leu ArgArg Leu Leu 805 810 815 Arg Asn Gly Glu Pro His Val Ser Ser Ala Lys ValVal Leu Val Asp 820 825 830 Gly Val Pro Gly Cys Gly Lys Thr Lys Glu IleLeu Ser Arg Val Asn 835 840 845 Phe Asp Glu Asp Leu Ile Leu Val Pro GlyLys Gln Ala Ala Glu Met 850 855 860 Ile Arg Arg Arg Ala Asn Ser Ser GlyIle Ile Val Ala Thr Lys Asp 865 870 875 880 Asn Val Lys Thr Val Asp SerPhe Met Met Asn Phe Gly Lys Ser Thr 885 890 895 Arg Cys Gln Phe Lys ArgLeu Phe Ile Asp Glu Gly Leu Met Leu His 900 905 910 Thr Gly Cys Val AsnPhe Leu Val Ala Met Ser Leu Cys Glu Ile Ala 915 920 925 Tyr Val Tyr GlyAsp Thr Gln Gln Ile Pro Tyr Ile Asn Arg Val Ser 930 935 940 Gly Phe ProTyr Pro Ala His Phe Ala Lys Leu Glu Val Asp Glu Val 945 950 955 960 GluThr Arg Arg Thr Thr Leu Arg Cys Pro Ala Asp Val Thr His Tyr 965 970 975Leu Asn Arg Arg Tyr Glu Gly Phe Val Met Ser Thr Ser Ser Val Lys 980 985990 Lys Ser Val Ser Gln Glu Met Val Gly Gly Ala Ala Val Ile Asn Pro 9951000 1005 Ile Ser Lys Pro Leu His Gly Lys Ile Leu Thr Phe Thr Gln Ser1010 1015 1020 Asp Lys Glu Ala Leu Leu Ser Arg Gly Tyr Ser Asp Val HisThr 1025 1030 1035 Val His Glu Val Gln Gly Glu Thr Tyr Ser Asp Val SerLeu Val 1040 1045 1050 Arg Leu Thr Pro Thr Pro Val Ser Ile Ile Ala GlyAsp Ser Pro 1055 1060 1065 His Val Leu Val Ala Leu Ser Arg His Thr CysSer Leu Lys Tyr 1070 1075 1080 Tyr Thr Val Val Met Asp Pro Leu Val SerIle Ile Arg Asp Leu 1085 1090 1095 Glu Lys Leu Ser Ser Tyr Leu Leu AspMet Tyr Lys Val Asp Ala 1100 1105 1110 Gly Thr Gln Xaa Gln Leu Gln IleAsp Ser Val Phe Lys Gly Ser 1115 1120 1125 Asn Leu Phe Val Ala Ala ProLys Thr Gly Asp Ile Ser Asp Met 1130 1135 1140 Gln Phe Tyr Tyr Asp LysCys Leu Pro Gly Asn Ser Thr Met Met 1145 1150 1155 Asn Asn Phe Asp AlaVal Thr Met Arg Leu Thr Asp Ile Ser Leu 1160 1165 1170 Asn Val Lys AspCys Ile Leu Asp Met Ser Lys Ser Val Ala Ala 1175 1180 1185 Pro Lys AspGln Ile Lys Pro Leu Ile Pro Met Val Arg Thr Ala 1190 1195 1200 Ala GluMet Pro Arg Gln Thr Gly Leu Leu Glu Asn Leu Val Ala 1205 1210 1215 MetIle Lys Arg Asn Phe Asn Ala Pro Glu Leu Ser Gly Ile Ile 1220 1225 1230Asp Ile Glu Asn Thr Ala Ser Leu Val Val Asp Lys Phe Phe Asp 1235 12401245 Ser Tyr Leu Leu Lys Glu Lys Arg Lys Pro Asn Lys Asn Val Ser 12501255 1260 Leu Phe Ser Arg Glu Ser Leu Asn Arg Trp Leu Glu Lys Gln Glu1265 1270 1275 Gln Val Thr Ile Gly Gln Leu Ala Asp Phe Asp Phe Val AspLeu 1280 1285 1290 Pro Ala Val Asp Gln Tyr Arg His Met Ile Lys Ala GlnPro Lys 1295 1300 1305 Gln Lys Leu Asp Thr Ser Ile Gln Thr Glu Tyr ProAla Leu Gln 1310 1315 1320 Thr Ile Val Tyr His Ser Lys Lys Ile Asn AlaIle Phe Gly Pro 1325 1330 1335 Leu Phe Ser Glu Leu Thr Arg Gln Leu LeuAsp Ser Val Asp Ser 1340 1345 1350 Ser Arg Phe Leu Phe Phe Thr Arg LysThr Pro Ala Gln Ile Glu 1355 1360 1365 Asp Phe Phe Gly Asp Leu Asp SerHis Val Pro Met Asp Val Leu 1370 1375 1380 Glu Leu Asp Ile Ser Lys TyrAsp Lys Ser Gln Asn Glu Phe His 1385 1390 1395 Cys Ala Val Glu Tyr GluIle Trp Arg Arg Leu Gly Phe Glu Asp 1400 1405 1410 Phe Leu Gly Glu ValTrp Lys Gln Gly His Arg Lys Thr Thr Leu 1415 1420 1425 Lys Asp Tyr ThrAla Gly Ile Lys Thr Cys Ile Trp Tyr Gln Arg 1430 1435 1440 Lys Ser GlyAsp Val Thr Thr Phe Ile Gly Asn Thr Val Ile Ile 1445 1450 1455 Ala AlaCys Leu Ala Ser Met Leu Pro Met Glu Lys Ile Ile Lys 1460 1465 1470 GlyAla Phe Cys Gly Asp Asp Ser Leu Leu Tyr Phe Pro Lys Gly 1475 1480 1485Cys Glu Phe Pro Asp Val Gln His Ser Ala Asn Leu Met Trp Asn 1490 14951500 Phe Glu Ala Lys Leu Phe Lys Lys Gln Tyr Gly Tyr Phe Cys Gly 15051510 1515 Arg Tyr Val Ile His His Asp Arg Gly Cys Ile Val Tyr Tyr Asp1520 1525 1530 Pro Leu Lys Leu Ile Ser Lys Leu Gly Ala Lys His Ile LysAsp 1535 1540 1545 Trp Glu His Leu Glu Glu Phe Arg Arg Ser Leu Cys AspVal Ala 1550 1555 1560 Val Ser Leu Asn Asn Cys Ala Tyr Tyr Thr Gln LeuAsp Asp Ala 1565 1570 1575 Val Trp Glu Val His Lys Thr Ala Pro Pro GlySer Phe Val Tyr 1580 1585 1590 Lys Ser Leu Val Lys Tyr Leu Ser Asp LysVal Leu Phe Arg Ser 1595 1600 1605 Leu Phe Ile Asp Gly Ser Ser Cys 16101615 5 3351 DNA Tobacco mosaic virus CDS (1)..(3348) misc_feature(1096)..(1096) n is “t”, “c”, “a” or “g”, except when nucleotide 1097 is“t” and nucleotide 1098 is “t” or “c”, n cannot be “t” 5 atg gca tac acacag aca gct acc aca tca gct ttg ctg gac act gtc 48 Met Ala Tyr Thr GlnThr Ala Thr Thr Ser Ala Leu Leu Asp Thr Val 1 5 10 15 cga gga aac aactcc ttg gtc aat gat cta gca aag cgt cgt ctt tac 96 Arg Gly Asn Asn SerLeu Val Asn Asp Leu Ala Lys Arg Arg Leu Tyr 20 25 30 gac aca gcg gtt gaagag ttt aac gct cgt gac cgc agg ccc aaa gtg 144 Asp Thr Ala Val Glu GluPhe Asn Ala Arg Asp Arg Arg Pro Lys Val 35 40 45 aac ttt tca aaa gta ataagc gag gag cag acg ctt att gct acc cgg 192 Asn Phe Ser Lys Val Ile SerGlu Glu Gln Thr Leu Ile Ala Thr Arg 50 55 60 gcg tat cca gaa ttc caa attaca ttt tat aac acg caa aat gcc gtg 240 Ala Tyr Pro Glu Phe Gln Ile ThrPhe Tyr Asn Thr Gln Asn Ala Val 65 70 75 80 cat tcg ctt gca ggt gga ttgcga tct tta gaa ctg gaa tat ctg atg 288 His Ser Leu Ala Gly Gly Leu ArgSer Leu Glu Leu Glu Tyr Leu Met 85 90 95 atg caa att ccc tac gga tca ttgact tat gac ata ggc ggg aat ttt 336 Met Gln Ile Pro Tyr Gly Ser Leu ThrTyr Asp Ile Gly Gly Asn Phe 100 105 110 gca tcg cat ctg ttc aag gga cgagca tat gta cac tgc tgc atg ccc 384 Ala Ser His Leu Phe Lys Gly Arg AlaTyr Val His Cys Cys Met Pro 115 120 125 aac ctg gac gtt cga gac atc atgcgg cat gaa ggc cag aaa gac agt 432 Asn Leu Asp Val Arg Asp Ile Met ArgHis Glu Gly Gln Lys Asp Ser 130 135 140 att gaa cta tac ctt tct agg ctagag aga ggg gga aaa aca gtc ccc 480 Ile Glu Leu Tyr Leu Ser Arg Leu GluArg Gly Gly Lys Thr Val Pro 145 150 155 160 aac ttc caa aag gaa gca tttgac aga tac gca gaa att cct gaa gac 528 Asn Phe Gln Lys Glu Ala Phe AspArg Tyr Ala Glu Ile Pro Glu Asp 165 170 175 gct gtc tgt cac aat act ttccag aca tgc gaa cat cag ccg atg caa 576 Ala Val Cys His Asn Thr Phe GlnThr Cys Glu His Gln Pro Met Gln 180 185 190 caa tca ggc aga gtg tat gccatt gcg cta cac agc ata tat gac ata 624 Gln Ser Gly Arg Val Tyr Ala IleAla Leu His Ser Ile Tyr Asp Ile 195 200 205 ccc gct gat gag ttc ggg gcagca ctc ttg agg aaa aat gtc cat acg 672 Pro Ala Asp Glu Phe Gly Ala AlaLeu Leu Arg Lys Asn Val His Thr 210 215 220 tgc tat gcc gct ttc cac ttctct gag aac ctg ctt ctt gaa gat tca 720 Cys Tyr Ala Ala Phe His Phe SerGlu Asn Leu Leu Leu Glu Asp Ser 225 230 235 240 tac gtc aat ctg gac gaaatc aac gcg tgt ttt tcg cgc gat gga gac 768 Tyr Val Asn Leu Asp Glu IleAsn Ala Cys Phe Ser Arg Asp Gly Asp 245 250 255 aag ttg acc ttt tct tttgca tca gag agt act ctt aat tac tgt cat 816 Lys Leu Thr Phe Ser Phe AlaSer Glu Ser Thr Leu Asn Tyr Cys His 260 265 270 agt tat tct aat att cttaag tat gtg tgc aaa act tac ttc ccg gcc 864 Ser Tyr Ser Asn Ile Leu LysTyr Val Cys Lys Thr Tyr Phe Pro Ala 275 280 285 tct aat aga gag gtt tacatg aag gag ttt tta gtc acc agg gtt aat 912 Ser Asn Arg Glu Val Tyr MetLys Glu Phe Leu Val Thr Arg Val Asn 290 295 300 acc tgg ttt tgt aag ttttct aga ata gat act ttt ctt ttg tac aaa 960 Thr Trp Phe Cys Lys Phe SerArg Ile Asp Thr Phe Leu Leu Tyr Lys 305 310 315 320 ggt gtg gcc cat aaaggt gta gat agt gag cag ttt tat act gca atg 1008 Gly Val Ala His Lys GlyVal Asp Ser Glu Gln Phe Tyr Thr Ala Met 325 330 335 gaa gac gca tgg cattac aaa aag act ctt gca atg tgc aac agc gag 1056 Glu Asp Ala Trp His TyrLys Lys Thr Leu Ala Met Cys Asn Ser Glu 340 345 350 aga atc ctc ctt gaggat tca tca aca gtc aat tac tgg nnn ccc gaa 1104 Arg Ile Leu Leu Glu AspSer Ser Thr Val Asn Tyr Trp Xaa Pro Glu 355 360 365 atg agg gat atg gtcatc gta cca tta ttc gac att tct ttg gag act 1152 Met Arg Asp Met Val IleVal Pro Leu Phe Asp Ile Ser Leu Glu Thr 370 375 380 agt aag agg acg cgcaag gaa gtc tta gtg tcc aag gat ttc gtg ttt 1200 Ser Lys Arg Thr Arg LysGlu Val Leu Val Ser Lys Asp Phe Val Phe 385 390 395 400 aca gtg ctt aaccac att cga aca tac cag gca aaa gct ctt aca tac 1248 Thr Val Leu Asn HisIle Arg Thr Tyr Gln Ala Lys Ala Leu Thr Tyr 405 410 415 gta aat gtt ttgtcc ttc gtc gaa tcg att cga tcg agg gta atc att 1296 Val Asn Val Leu SerPhe Val Glu Ser Ile Arg Ser Arg Val Ile Ile 420 425 430 aac ggt gtg acagcg agg tcc gaa tgg gat gtg gac aaa tct ttg tta 1344 Asn Gly Val Thr AlaArg Ser Glu Trp Asp Val Asp Lys Ser Leu Leu 435 440 445 caa tcc ttg tccatg acg ttt tac ctg cat act aag ctt gcc gtt cta 1392 Gln Ser Leu Ser MetThr Phe Tyr Leu His Thr Lys Leu Ala Val Leu 450 455 460 aag gat gac ttactg att agc aag ttt agt ctc ggt tcg aaa acg gtg 1440 Lys Asp Asp Leu LeuIle Ser Lys Phe Ser Leu Gly Ser Lys Thr Val 465 470 475 480 tgc cag catgtg tgg gat gag att tca ctg gcg ttt ggg aac gca ttt 1488 Cys Gln His ValTrp Asp Glu Ile Ser Leu Ala Phe Gly Asn Ala Phe 485 490 495 ccc tcc gtgaaa gag agg ctc ttg aac agg aaa ctt atc aga gtg gca 1536 Pro Ser Val LysGlu Arg Leu Leu Asn Arg Lys Leu Ile Arg Val Ala 500 505 510 ggc gac gcacta gag atc agg gtg cct gat cta tat gtg acc ttc cac 1584 Gly Asp Ala LeuGlu Ile Arg Val Pro Asp Leu Tyr Val Thr Phe His 515 520 525 gac cga ttagtg act gag tac aag gcc tct gtg gac atg cct gcg ctt 1632 Asp Arg Leu ValThr Glu Tyr Lys Ala Ser Val Asp Met Pro Ala Leu 530 535 540 gac att aggaag aag atg gaa gaa acg gaa gtg atg tac aat gca ctt 1680 Asp Ile Arg LysLys Met Glu Glu Thr Glu Val Met Tyr Asn Ala Leu 545 550 555 560 tca gagtta tcg gtg tta agg gag tct gac aaa ttc gat gtt gat gtt 1728 Ser Glu LeuSer Val Leu Arg Glu Ser Asp Lys Phe Asp Val Asp Val 565 570 575 ttt tcccag atg tgc caa tct ttg gaa gtt gac gca atg acg gca gcg 1776 Phe Ser GlnMet Cys Gln Ser Leu Glu Val Asp Ala Met Thr Ala Ala 580 585 590 aag gttata gtc gcg gtc atg agc aat aag agc ggt ctg act ctc aca 1824 Lys Val IleVal Ala Val Met Ser Asn Lys Ser Gly Leu Thr Leu Thr 595 600 605 ttt gaacga cct act gag gcg aat gtt gcg cta gct tta cag gat caa 1872 Phe Glu ArgPro Thr Glu Ala Asn Val Ala Leu Ala Leu Gln Asp Gln 610 615 620 gaa aaggct tca gaa ggt gct ttg gta gtt acc tca aga gaa gtt gaa 1920 Glu Lys AlaSer Glu Gly Ala Leu Val Val Thr Ser Arg Glu Val Glu 625 630 635 640 gaaccg tcc atg aag ggt tcg atg gcc aga gga gag tta caa tta gct 1968 Glu ProSer Met Lys Gly Ser Met Ala Arg Gly Glu Leu Gln Leu Ala 645 650 655 ggtctt gct gga gat cat ccg gag tcg tcc tat tct agg aac gag gag 2016 Gly LeuAla Gly Asp His Pro Glu Ser Ser Tyr Ser Arg Asn Glu Glu 660 665 670 atagag tct tta gag cag ttt cat atg gca acg gca gat tcg tta att 2064 Ile GluSer Leu Glu Gln Phe His Met Ala Thr Ala Asp Ser Leu Ile 675 680 685 cgtaag cag atg agc tcg att gtg tac acg ggt ccg att aaa gtt cag 2112 Arg LysGln Met Ser Ser Ile Val Tyr Thr Gly Pro Ile Lys Val Gln 690 695 700 caaatg aaa aac ttt atc gat agc ctg gta gca tca cta tct gct gcg 2160 Gln MetLys Asn Phe Ile Asp Ser Leu Val Ala Ser Leu Ser Ala Ala 705 710 715 720gtg tcg aat ctc gtc aag atc ctc aaa gat aca gct gct att gac ctt 2208 ValSer Asn Leu Val Lys Ile Leu Lys Asp Thr Ala Ala Ile Asp Leu 725 730 735gaa acc cgt caa aag ttt gga gtc ttg gat gtt aca tct agg aag tgg 2256 GluThr Arg Gln Lys Phe Gly Val Leu Asp Val Thr Ser Arg Lys Trp 740 745 750tta att aaa cca acg gcc aag agt cat gca tgg ggt gtt gtt gaa acc 2304 LeuIle Lys Pro Thr Ala Lys Ser His Ala Trp Gly Val Val Glu Thr 755 760 765cac gcg agg aag tat cat gtg gcg ctt ctg gaa tat gat gag cag ggt 2352 HisAla Arg Lys Tyr His Val Ala Leu Leu Glu Tyr Asp Glu Gln Gly 770 775 780gtg gtg aca tgc gat gat tgg aga aga gta gct gtc agc tct gag tct 2400 ValVal Thr Cys Asp Asp Trp Arg Arg Val Ala Val Ser Ser Glu Ser 785 790 795800 gtt gtt tat tcc gac atg gcg aaa ctc aga act ctg cgc aga ctg ctt 2448Val Val Tyr Ser Asp Met Ala Lys Leu Arg Thr Leu Arg Arg Leu Leu 805 810815 cga aac gga gaa ccg cat gtc agt agc gca aag gtt gtt ctt gtg gac 2496Arg Asn Gly Glu Pro His Val Ser Ser Ala Lys Val Val Leu Val Asp 820 825830 gga gtt ccg ggc tgt gga aaa acc aaa gaa att ctt tcc agg gtt aat 2544Gly Val Pro Gly Cys Gly Lys Thr Lys Glu Ile Leu Ser Arg Val Asn 835 840845 ttt gat gaa gat cta att tta gta cct ggg aag caa gct gct gaa atg 2592Phe Asp Glu Asp Leu Ile Leu Val Pro Gly Lys Gln Ala Ala Glu Met 850 855860 atc aga aga cgt gcg aat tcc tca ggg att att gtg gcc acg aag gac 2640Ile Arg Arg Arg Ala Asn Ser Ser Gly Ile Ile Val Ala Thr Lys Asp 865 870875 880 aac gtt aaa acc gtt gat tct ttc atg atg aat ttt ggg aaa agc aca2688 Asn Val Lys Thr Val Asp Ser Phe Met Met Asn Phe Gly Lys Ser Thr 885890 895 cgc tgt cag ttc aag agg tta ttc att gat gaa ggg ttg atg ttg cat2736 Arg Cys Gln Phe Lys Arg Leu Phe Ile Asp Glu Gly Leu Met Leu His 900905 910 act ggt tgt gtt aat ttt ctt gtg gcg atg tca ttg tgc gaa att gca2784 Thr Gly Cys Val Asn Phe Leu Val Ala Met Ser Leu Cys Glu Ile Ala 915920 925 tat gtt tac gga gac aca cag cag att cca tac atc aat aga gtt tca2832 Tyr Val Tyr Gly Asp Thr Gln Gln Ile Pro Tyr Ile Asn Arg Val Ser 930935 940 gga ttc ccg tac ccc gcc cat ttt gcc aaa ttg gaa gtt gac gag gtg2880 Gly Phe Pro Tyr Pro Ala His Phe Ala Lys Leu Glu Val Asp Glu Val 945950 955 960 gag aca cgc aga act act ctc cgt tgt cca gcc gat gtc aca cattat 2928 Glu Thr Arg Arg Thr Thr Leu Arg Cys Pro Ala Asp Val Thr His Tyr965 970 975 ctg aac agg aga tat gag ggc ttt gtc atg agc act tct tcg gttaaa 2976 Leu Asn Arg Arg Tyr Glu Gly Phe Val Met Ser Thr Ser Ser Val Lys980 985 990 aag tct gtt tcg cag gag atg gtc ggc gga gcc gcc gtg atc aatccg 3024 Lys Ser Val Ser Gln Glu Met Val Gly Gly Ala Ala Val Ile Asn Pro995 1000 1005 atc tca aaa ccc ttg cat ggc aag atc ctg act ttt acc caatcg 3069 Ile Ser Lys Pro Leu His Gly Lys Ile Leu Thr Phe Thr Gln Ser1010 1015 1020 gat aaa gaa gct ctg ctt tca aga ggg tat tca gat gtt cacact 3114 Asp Lys Glu Ala Leu Leu Ser Arg Gly Tyr Ser Asp Val His Thr1025 1030 1035 gtg cat gaa gtg caa ggc gag aca tac tct gat gtt tca ctagtt 3159 Val His Glu Val Gln Gly Glu Thr Tyr Ser Asp Val Ser Leu Val1040 1045 1050 agg cta acc cct aca cca gtc tcc atc att gca gga gac agcccg 3204 Arg Leu Thr Pro Thr Pro Val Ser Ile Ile Ala Gly Asp Ser Pro1055 1060 1065 cat gtt ttg gtc gca ttg tca agg cac acc tgt tcg ctc aagtac 3249 His Val Leu Val Ala Leu Ser Arg His Thr Cys Ser Leu Lys Tyr1070 1075 1080 tac act gtt gtt atg gat cct tta gtt agt atc att aga gatcta 3294 Tyr Thr Val Val Met Asp Pro Leu Val Ser Ile Ile Arg Asp Leu1085 1090 1095 gag aaa ctt agc tcg tac ttg tta gat atg tat aag gtc gatgca 3339 Glu Lys Leu Ser Ser Tyr Leu Leu Asp Met Tyr Lys Val Asp Ala1100 1105 1110 gga aca caa tag 3351 Gly Thr Gln 1115 6 1116 PRT Tobaccomosaic virus misc_feature (366)..(366) The ′Xaa′ at location 366 standsfor any amino acid except Phe. 6 Met Ala Tyr Thr Gln Thr Ala Thr Thr SerAla Leu Leu Asp Thr Val 1 5 10 15 Arg Gly Asn Asn Ser Leu Val Asn AspLeu Ala Lys Arg Arg Leu Tyr 20 25 30 Asp Thr Ala Val Glu Glu Phe Asn AlaArg Asp Arg Arg Pro Lys Val 35 40 45 Asn Phe Ser Lys Val Ile Ser Glu GluGln Thr Leu Ile Ala Thr Arg 50 55 60 Ala Tyr Pro Glu Phe Gln Ile Thr PheTyr Asn Thr Gln Asn Ala Val 65 70 75 80 His Ser Leu Ala Gly Gly Leu ArgSer Leu Glu Leu Glu Tyr Leu Met 85 90 95 Met Gln Ile Pro Tyr Gly Ser LeuThr Tyr Asp Ile Gly Gly Asn Phe 100 105 110 Ala Ser His Leu Phe Lys GlyArg Ala Tyr Val His Cys Cys Met Pro 115 120 125 Asn Leu Asp Val Arg AspIle Met Arg His Glu Gly Gln Lys Asp Ser 130 135 140 Ile Glu Leu Tyr LeuSer Arg Leu Glu Arg Gly Gly Lys Thr Val Pro 145 150 155 160 Asn Phe GlnLys Glu Ala Phe Asp Arg Tyr Ala Glu Ile Pro Glu Asp 165 170 175 Ala ValCys His Asn Thr Phe Gln Thr Cys Glu His Gln Pro Met Gln 180 185 190 GlnSer Gly Arg Val Tyr Ala Ile Ala Leu His Ser Ile Tyr Asp Ile 195 200 205Pro Ala Asp Glu Phe Gly Ala Ala Leu Leu Arg Lys Asn Val His Thr 210 215220 Cys Tyr Ala Ala Phe His Phe Ser Glu Asn Leu Leu Leu Glu Asp Ser 225230 235 240 Tyr Val Asn Leu Asp Glu Ile Asn Ala Cys Phe Ser Arg Asp GlyAsp 245 250 255 Lys Leu Thr Phe Ser Phe Ala Ser Glu Ser Thr Leu Asn TyrCys His 260 265 270 Ser Tyr Ser Asn Ile Leu Lys Tyr Val Cys Lys Thr TyrPhe Pro Ala 275 280 285 Ser Asn Arg Glu Val Tyr Met Lys Glu Phe Leu ValThr Arg Val Asn 290 295 300 Thr Trp Phe Cys Lys Phe Ser Arg Ile Asp ThrPhe Leu Leu Tyr Lys 305 310 315 320 Gly Val Ala His Lys Gly Val Asp SerGlu Gln Phe Tyr Thr Ala Met 325 330 335 Glu Asp Ala Trp His Tyr Lys LysThr Leu Ala Met Cys Asn Ser Glu 340 345 350 Arg Ile Leu Leu Glu Asp SerSer Thr Val Asn Tyr Trp Xaa Pro Glu 355 360 365 Met Arg Asp Met Val IleVal Pro Leu Phe Asp Ile Ser Leu Glu Thr 370 375 380 Ser Lys Arg Thr ArgLys Glu Val Leu Val Ser Lys Asp Phe Val Phe 385 390 395 400 Thr Val LeuAsn His Ile Arg Thr Tyr Gln Ala Lys Ala Leu Thr Tyr 405 410 415 Val AsnVal Leu Ser Phe Val Glu Ser Ile Arg Ser Arg Val Ile Ile 420 425 430 AsnGly Val Thr Ala Arg Ser Glu Trp Asp Val Asp Lys Ser Leu Leu 435 440 445Gln Ser Leu Ser Met Thr Phe Tyr Leu His Thr Lys Leu Ala Val Leu 450 455460 Lys Asp Asp Leu Leu Ile Ser Lys Phe Ser Leu Gly Ser Lys Thr Val 465470 475 480 Cys Gln His Val Trp Asp Glu Ile Ser Leu Ala Phe Gly Asn AlaPhe 485 490 495 Pro Ser Val Lys Glu Arg Leu Leu Asn Arg Lys Leu Ile ArgVal Ala 500 505 510 Gly Asp Ala Leu Glu Ile Arg Val Pro Asp Leu Tyr ValThr Phe His 515 520 525 Asp Arg Leu Val Thr Glu Tyr Lys Ala Ser Val AspMet Pro Ala Leu 530 535 540 Asp Ile Arg Lys Lys Met Glu Glu Thr Glu ValMet Tyr Asn Ala Leu 545 550 555 560 Ser Glu Leu Ser Val Leu Arg Glu SerAsp Lys Phe Asp Val Asp Val 565 570 575 Phe Ser Gln Met Cys Gln Ser LeuGlu Val Asp Ala Met Thr Ala Ala 580 585 590 Lys Val Ile Val Ala Val MetSer Asn Lys Ser Gly Leu Thr Leu Thr 595 600 605 Phe Glu Arg Pro Thr GluAla Asn Val Ala Leu Ala Leu Gln Asp Gln 610 615 620 Glu Lys Ala Ser GluGly Ala Leu Val Val Thr Ser Arg Glu Val Glu 625 630 635 640 Glu Pro SerMet Lys Gly Ser Met Ala Arg Gly Glu Leu Gln Leu Ala 645 650 655 Gly LeuAla Gly Asp His Pro Glu Ser Ser Tyr Ser Arg Asn Glu Glu 660 665 670 IleGlu Ser Leu Glu Gln Phe His Met Ala Thr Ala Asp Ser Leu Ile 675 680 685Arg Lys Gln Met Ser Ser Ile Val Tyr Thr Gly Pro Ile Lys Val Gln 690 695700 Gln Met Lys Asn Phe Ile Asp Ser Leu Val Ala Ser Leu Ser Ala Ala 705710 715 720 Val Ser Asn Leu Val Lys Ile Leu Lys Asp Thr Ala Ala Ile AspLeu 725 730 735 Glu Thr Arg Gln Lys Phe Gly Val Leu Asp Val Thr Ser ArgLys Trp 740 745 750 Leu Ile Lys Pro Thr Ala Lys Ser His Ala Trp Gly ValVal Glu Thr 755 760 765 His Ala Arg Lys Tyr His Val Ala Leu Leu Glu TyrAsp Glu Gln Gly 770 775 780 Val Val Thr Cys Asp Asp Trp Arg Arg Val AlaVal Ser Ser Glu Ser 785 790 795 800 Val Val Tyr Ser Asp Met Ala Lys LeuArg Thr Leu Arg Arg Leu Leu 805 810 815 Arg Asn Gly Glu Pro His Val SerSer Ala Lys Val Val Leu Val Asp 820 825 830 Gly Val Pro Gly Cys Gly LysThr Lys Glu Ile Leu Ser Arg Val Asn 835 840 845 Phe Asp Glu Asp Leu IleLeu Val Pro Gly Lys Gln Ala Ala Glu Met 850 855 860 Ile Arg Arg Arg AlaAsn Ser Ser Gly Ile Ile Val Ala Thr Lys Asp 865 870 875 880 Asn Val LysThr Val Asp Ser Phe Met Met Asn Phe Gly Lys Ser Thr 885 890 895 Arg CysGln Phe Lys Arg Leu Phe Ile Asp Glu Gly Leu Met Leu His 900 905 910 ThrGly Cys Val Asn Phe Leu Val Ala Met Ser Leu Cys Glu Ile Ala 915 920 925Tyr Val Tyr Gly Asp Thr Gln Gln Ile Pro Tyr Ile Asn Arg Val Ser 930 935940 Gly Phe Pro Tyr Pro Ala His Phe Ala Lys Leu Glu Val Asp Glu Val 945950 955 960 Glu Thr Arg Arg Thr Thr Leu Arg Cys Pro Ala Asp Val Thr HisTyr 965 970 975 Leu Asn Arg Arg Tyr Glu Gly Phe Val Met Ser Thr Ser SerVal Lys 980 985 990 Lys Ser Val Ser Gln Glu Met Val Gly Gly Ala Ala ValIle Asn Pro 995 1000 1005 Ile Ser Lys Pro Leu His Gly Lys Ile Leu ThrPhe Thr Gln Ser 1010 1015 1020 Asp Lys Glu Ala Leu Leu Ser Arg Gly TyrSer Asp Val His Thr 1025 1030 1035 Val His Glu Val Gln Gly Glu Thr TyrSer Asp Val Ser Leu Val 1040 1045 1050 Arg Leu Thr Pro Thr Pro Val SerIle Ile Ala Gly Asp Ser Pro 1055 1060 1065 His Val Leu Val Ala Leu SerArg His Thr Cys Ser Leu Lys Tyr 1070 1075 1080 Tyr Thr Val Val Met AspPro Leu Val Ser Ile Ile Arg Asp Leu 1085 1090 1095 Glu Lys Leu Ser SerTyr Leu Leu Asp Met Tyr Lys Val Asp Ala 1100 1105 1110 Gly Thr Gln 11157 4834 DNA Tobacco mosaic virus misc_feature (1096)..(1096) n is “t”,“c”, “a” or “g”, except when nucleotide 1097 is “t” and nucleotide 1098is “t” or “c”, n cannot be “t” 7 atggcataca cacagacagc taccacatcagctttgctgg acactgtccg aggaaacaac 60 tccttggtca atgatctagc aaagcgtcgtctttacgaca cagcggttga agagtttaac 120 gctcgtgacc gcaggcccaa agtgaacttttcaaaagtaa taagcgagga gcagacgctt 180 attgctaccc gggcgtatcc agaattccaaattacatttt ataacacgca aaatgccgtg 240 cattcgcttg caggtggatt gcgatctttagaactggaat atctgatgat gcaaattccc 300 tacggatcat tgacttatga cataggcgggaattttgcat cgcatctgtt caagggacga 360 gcatatgtac actgctgcat gcccaacctggacgttcgag acatcatgcg gcatgaaggc 420 cagaaagaca gtattgaact atacctttctaggctagaga gagggggaaa aacagtcccc 480 aacttccaaa aggaagcatt tgacagatacgcagaaattc ctgaagacgc tgtctgtcac 540 aatactttcc agacatgcga acatcagccgatgcaacaat caggcagagt gtatgccatt 600 gcgctacaca gcatatatga catacccgctgatgagttcg gggcagcact cttgaggaaa 660 aatgtccata cgtgctatgc cgctttccacttctctgaga acctgcttct tgaagattca 720 tacgtcaatc tggacgaaat caacgcgtgtttttcgcgcg atggagacaa gttgaccttt 780 tcttttgcat cagagagtac tcttaattactgtcatagtt attctaatat tcttaagtat 840 gtgtgcaaaa cttacttccc ggcctctaatagagaggttt acatgaagga gtttttagtc 900 accagggtta atacctggtt ttgtaagttttctagaatag atacttttct tttgtacaaa 960 ggtgtggccc ataaaggtgt agatagtgagcagttttata ctgcaatgga agacgcatgg 1020 cattacaaaa agactcttgc aatgtgcaacagcgagagaa tcctccttga ggattcatca 1080 acagtcaatt actggnnncc cgaaatgagggatatggtca tcgtaccatt attcgacatt 1140 tctttggaga ctagtaagag gacgcgcaaggaagtcttag tgtccaagga tttcgtgttt 1200 acagtgctta accacattcg aacataccaggcaaaagctc ttacatacgt aaatgttttg 1260 tccttcgtcg aatcgattcg atcgagggtaatcattaacg gtgtgacagc gaggtccgaa 1320 tgggatgtgg acaaatcttt gttacaatccttgtccatga cgttttacct gcatactaag 1380 cttgccgttc taaaggatga cttactgattagcaagttta gtctcggttc gaaaacggtg 1440 tgccagcatg tgtgggatga gatttcactggcgtttggga acgcatttcc ctccgtgaaa 1500 gagaggctct tgaacaggaa acttatcagagtggcaggcg acgcactaga gatcagggtg 1560 cctgatctat atgtgacctt ccacgaccgattagtgactg agtacaaggc ctctgtggac 1620 atgcctgcgc ttgacattag gaagaagatggaagaaacgg aagtgatgta caatgcactt 1680 tcagagttat cggtgttaag ggagtctgacaaattcgatg ttgatgtttt ttcccagatg 1740 tgccaatctt tggaagttga cgcaatgacggcagcgaagg ttatagtcgc ggtcatgagc 1800 aataagagcg gtctgactct cacatttgaacgacctactg aggcgaatgt tgcgctagct 1860 ttacaggatc aagaaaaggc ttcagaaggtgctttggtag ttacctcaag agaagttgaa 1920 gaaccgtcca tgaagggttc gatggccagaggagagttac aattagctgg tcttgctgga 1980 gatcatccgg agtcgtccta ttctaggaacgaggagatag agtctttaga gcagtttcat 2040 atggcaacgg cagattcgtt aattcgtaagcagatgagct cgattgtgta cacgggtccg 2100 attaaagttc agcaaatgaa aaactttatcgatagcctgg tagcatcact atctgctgcg 2160 gtgtcgaatc tcgtcaagat cctcaaagatacagctgcta ttgaccttga aacccgtcaa 2220 aagtttggag tcttggatgt tacatctaggaagtggttaa ttaaaccaac ggccaagagt 2280 catgcatggg gtgttgttga aacccacgcgaggaagtatc atgtggcgct tctggaatat 2340 gatgagcagg gtgtggtgac atgcgatgattggagaagag tagctgtcag ctctgagtct 2400 gttgtttatt ccgacatggc gaaactcagaactctgcgca gactgcttcg aaacggagaa 2460 ccgcatgtca gtagcgcaaa ggttgttcttgtggacggag ttccgggctg tggaaaaacc 2520 aaagaaattc tttccagggt taattttgatgaagatctaa ttttagtacc tgggaagcaa 2580 gctgctgaaa tgatcagaag acgtgcgaattcctcaggga ttattgtggc cacgaaggac 2640 aacgttaaaa ccgttgattc tttcatgatgaattttggga aaagcacacg ctgtcagttc 2700 aagaggttat tcattgatga agggttgatgttgcatactg gttgtgttaa ttttcttgtg 2760 gcgatgtcat tgtgcgaaat tgcatatgtttacggagaca cacagcagat tccatacatc 2820 aatagagttt caggattccc gtaccccgcccattttgcca aattggaagt tgacgaggtg 2880 gagacacgca gaactactct ccgttgtccagccgatgtca cacattatct gaacaggaga 2940 tatgagggct ttgtcatgag cacttcttcggttaaaaagt ctgtttcgca ggagatggtc 3000 ggcggagccg ccgtgatcaa tccgatctcaaaacccttgc atggcaagat cctgactttt 3060 acccaatcgg ataaagaagc tctgctttcaagagggtatt cagatgttca cactgtgcat 3120 gaagtgcaag gcgagacata ctctgatgtttcactagtta ggctaacccc tacaccagtc 3180 tccatcattg caggagacag cccgcatgttttggtcgcat tgtcaaggca cacctgttcg 3240 ctcaagtact acactgttgt tatggatcctttagttagta tcattagaga tctagagaaa 3300 cttagctcgt acttgttaga tatgtataaggtcgatgcag gaacacaata gcaattacag 3360 attgactcgg tgttcaaagg ttccaatctttttgtggcag cgccaaagac tggtgatatt 3420 tctgatatgc agttttacta tgataagtgtctcccaggca acagcaccat gatgaataat 3480 tttgatgctg ttaccatgag gttgactgacatttcattga atgtcaaaga ttgcatattg 3540 gatatgtcta agtctgttgc tgcgcctaaggatcaaatca aaccactaat acctatggta 3600 cgaacggcgg cagaaatgcc acgccagactggactattgg aaaatttagt ggcgatgatt 3660 aaaaggaact ttaacgcacc cgagttgtctggcatcattg atattgaaaa tactgcatct 3720 ttagttgtag ataagttttt cgatagttatttgcttaaag aaaaaagaaa accaaataaa 3780 aatgtttctt tgttcagtag agagtctctcaatagatggt tagaaaagca ggaacaggta 3840 acaataggcc agctcgcaga ttttgattttgtagatttgc cagcagttga tcagtacaga 3900 cacatgatca aagcacaacc caagcaaaaattggacactt caatccaaac ggagtacccg 3960 gctttgcaga cgattgtgta ccattcgaaaaagatcaatg caatatttgg cccgttgttt 4020 agtgagctta ctaggcaatt actggacagtgttgattcga gcagattttt gtttttcaca 4080 agaaagacac cagcgcagat tgaggatttcttcggagatc tcgacagtca tgtgccgatg 4140 gatgtcttgg agctggatat atcaaaatacgacaaatctc agaatgaatt ccactgtgca 4200 gtagaatacg agatttggcg aagattgggttttgaagact tcttgggaga agtttggaaa 4260 caagggcata gaaagaccac cctcaaggattataccgcag gtatcaaaac ttgcatctgg 4320 tatcaaagaa agagtgggga cgtcacgacattcattggaa acactgtgat cattgctgca 4380 tgtttggcct cgatgcttcc gatggagaaaataatcaaag gagccttttg tggtgacgat 4440 agtctgctgt acttcccaaa gggttgtgagtttccggatg tgcaacactc cgcgaatctt 4500 atgtggaatt ttgaagcaaa actgtttaaaaaacagtatg gatacttttg cggaagatat 4560 gtaatacatc acgacagagg atgcattgtgtattacgatc ccctaaagtt gatctcgaaa 4620 cttggcgcta aacacatcaa ggattgggaacacttggagg agttcagaag gtctctttgt 4680 gatgttgctg tttcgttgaa caattgtgcgtattatacac agttggacga cgctgtatgg 4740 gaggttcata agaccgcccc tccaggttcgtttgtttata aaagtctggt gaagtatttg 4800 tctgataaag ttctttttag aagtttgtttatag 4834 8 1616 PRT Tobacco mosaic virus MISC_FEATURE (366)..(366) The′Xaa′ at location 366 stands for any amino acid except Phe. 8 Met AlaTyr Thr Gln Thr Ala Thr Thr Ser Ala Leu Leu Asp Thr Val 1 5 10 15 ArgGly Asn Asn Ser Leu Val Asn Asp Leu Ala Lys Arg Arg Leu Tyr 20 25 30 AspThr Ala Val Glu Glu Phe Asn Ala Arg Asp Arg Arg Pro Lys Val 35 40 45 AsnPhe Ser Lys Val Ile Ser Glu Glu Gln Thr Leu Ile Ala Thr Arg 50 55 60 AlaTyr Pro Glu Phe Gln Ile Thr Phe Tyr Asn Thr Gln Asn Ala Val 65 70 75 80His Ser Leu Ala Gly Gly Leu Arg Ser Leu Glu Leu Glu Tyr Leu Met 85 90 95Met Gln Ile Pro Tyr Gly Ser Leu Thr Tyr Asp Ile Gly Gly Asn Phe 100 105110 Ala Ser His Leu Phe Lys Gly Arg Ala Tyr Val His Cys Cys Met Pro 115120 125 Asn Leu Asp Val Arg Asp Ile Met Arg His Glu Gly Gln Lys Asp Ser130 135 140 Ile Glu Leu Tyr Leu Ser Arg Leu Glu Arg Gly Gly Lys Thr ValPro 145 150 155 160 Asn Phe Gln Lys Glu Ala Phe Asp Arg Tyr Ala Glu IlePro Glu Asp 165 170 175 Ala Val Cys His Asn Thr Phe Gln Thr Cys Glu HisGln Pro Met Gln 180 185 190 Gln Ser Gly Arg Val Tyr Ala Ile Ala Leu HisSer Ile Tyr Asp Ile 195 200 205 Pro Ala Asp Glu Phe Gly Ala Ala Leu LeuArg Lys Asn Val His Thr 210 215 220 Cys Tyr Ala Ala Phe His Phe Ser GluAsn Leu Leu Leu Glu Asp Ser 225 230 235 240 Tyr Val Asn Leu Asp Glu IleAsn Ala Cys Phe Ser Arg Asp Gly Asp 245 250 255 Lys Leu Thr Phe Ser PheAla Ser Glu Ser Thr Leu Asn Tyr Cys His 260 265 270 Ser Tyr Ser Asn IleLeu Lys Tyr Val Cys Lys Thr Tyr Phe Pro Ala 275 280 285 Ser Asn Arg GluVal Tyr Met Lys Glu Phe Leu Val Thr Arg Val Asn 290 295 300 Thr Trp PheCys Lys Phe Ser Arg Ile Asp Thr Phe Leu Leu Tyr Lys 305 310 315 320 GlyVal Ala His Lys Gly Val Asp Ser Glu Gln Phe Tyr Thr Ala Met 325 330 335Glu Asp Ala Trp His Tyr Lys Lys Thr Leu Ala Met Cys Asn Ser Glu 340 345350 Arg Ile Leu Leu Glu Asp Ser Ser Thr Val Asn Tyr Trp Xaa Pro Glu 355360 365 Met Arg Asp Met Val Ile Val Pro Leu Phe Asp Ile Ser Leu Glu Thr370 375 380 Ser Lys Arg Thr Arg Lys Glu Val Leu Val Ser Lys Asp Phe ValPhe 385 390 395 400 Thr Val Leu Asn His Ile Arg Thr Tyr Gln Ala Lys AlaLeu Thr Tyr 405 410 415 Val Asn Val Leu Ser Phe Val Glu Ser Ile Arg SerArg Val Ile Ile 420 425 430 Asn Gly Val Thr Ala Arg Ser Glu Trp Asp ValAsp Lys Ser Leu Leu 435 440 445 Gln Ser Leu Ser Met Thr Phe Tyr Leu HisThr Lys Leu Ala Val Leu 450 455 460 Lys Asp Asp Leu Leu Ile Ser Lys PheSer Leu Gly Ser Lys Thr Val 465 470 475 480 Cys Gln His Val Trp Asp GluIle Ser Leu Ala Phe Gly Asn Ala Phe 485 490 495 Pro Ser Val Lys Glu ArgLeu Leu Asn Arg Lys Leu Ile Arg Val Ala 500 505 510 Gly Asp Ala Leu GluIle Arg Val Pro Asp Leu Tyr Val Thr Phe His 515 520 525 Asp Arg Leu ValThr Glu Tyr Lys Ala Ser Val Asp Met Pro Ala Leu 530 535 540 Asp Ile ArgLys Lys Met Glu Glu Thr Glu Val Met Tyr Asn Ala Leu 545 550 555 560 SerGlu Leu Ser Val Leu Arg Glu Ser Asp Lys Phe Asp Val Asp Val 565 570 575Phe Ser Gln Met Cys Gln Ser Leu Glu Val Asp Ala Met Thr Ala Ala 580 585590 Lys Val Ile Val Ala Val Met Ser Asn Lys Ser Gly Leu Thr Leu Thr 595600 605 Phe Glu Arg Pro Thr Glu Ala Asn Val Ala Leu Ala Leu Gln Asp Gln610 615 620 Glu Lys Ala Ser Glu Gly Ala Leu Val Val Thr Ser Arg Glu ValGlu 625 630 635 640 Glu Pro Ser Met Lys Gly Ser Met Ala Arg Gly Glu LeuGln Leu Ala 645 650 655 Gly Leu Ala Gly Asp His Pro Glu Ser Ser Tyr SerArg Asn Glu Glu 660 665 670 Ile Glu Ser Leu Glu Gln Phe His Met Ala ThrAla Asp Ser Leu Ile 675 680 685 Arg Lys Gln Met Ser Ser Ile Val Tyr ThrGly Pro Ile Lys Val Gln 690 695 700 Gln Met Lys Asn Phe Ile Asp Ser LeuVal Ala Ser Leu Ser Ala Ala 705 710 715 720 Val Ser Asn Leu Val Lys IleLeu Lys Asp Thr Ala Ala Ile Asp Leu 725 730 735 Glu Thr Arg Gln Lys PheGly Val Leu Asp Val Thr Ser Arg Lys Trp 740 745 750 Leu Ile Lys Pro ThrAla Lys Ser His Ala Trp Gly Val Val Glu Thr 755 760 765 His Ala Arg LysTyr His Val Ala Leu Leu Glu Tyr Asp Glu Gln Gly 770 775 780 Val Val ThrCys Asp Asp Trp Arg Arg Val Ala Val Ser Ser Glu Ser 785 790 795 800 ValVal Tyr Ser Asp Met Ala Lys Leu Arg Thr Leu Arg Arg Leu Leu 805 810 815Arg Asn Gly Glu Pro His Val Ser Ser Ala Lys Val Val Leu Val Asp 820 825830 Gly Val Pro Gly Cys Gly Lys Thr Lys Glu Ile Leu Ser Arg Val Asn 835840 845 Phe Asp Glu Asp Leu Ile Leu Val Pro Gly Lys Gln Ala Ala Glu Met850 855 860 Ile Arg Arg Arg Ala Asn Ser Ser Gly Ile Ile Val Ala Thr LysAsp 865 870 875 880 Asn Val Lys Thr Val Asp Ser Phe Met Met Asn Phe GlyLys Ser Thr 885 890 895 Arg Cys Gln Phe Lys Arg Leu Phe Ile Asp Glu GlyLeu Met Leu His 900 905 910 Thr Gly Cys Val Asn Phe Leu Val Ala Met SerLeu Cys Glu Ile Ala 915 920 925 Tyr Val Tyr Gly Asp Thr Gln Gln Ile ProTyr Ile Asn Arg Val Ser 930 935 940 Gly Phe Pro Tyr Pro Ala His Phe AlaLys Leu Glu Val Asp Glu Val 945 950 955 960 Glu Thr Arg Arg Thr Thr LeuArg Cys Pro Ala Asp Val Thr His Tyr 965 970 975 Leu Asn Arg Arg Tyr GluGly Phe Val Met Ser Thr Ser Ser Val Lys 980 985 990 Lys Ser Val Ser GlnGlu Met Val Gly Gly Ala Ala Val Ile Asn Pro 995 1000 1005 Ile Ser LysPro Leu His Gly Lys Ile Leu Thr Phe Thr Gln Ser 1010 1015 1020 Asp LysGlu Ala Leu Leu Ser Arg Gly Tyr Ser Asp Val His Thr 1025 1030 1035 ValHis Glu Val Gln Gly Glu Thr Tyr Ser Asp Val Ser Leu Val 1040 1045 1050Arg Leu Thr Pro Thr Pro Val Ser Ile Ile Ala Gly Asp Ser Pro 1055 10601065 His Val Leu Val Ala Leu Ser Arg His Thr Cys Ser Leu Lys Tyr 10701075 1080 Tyr Thr Val Val Met Asp Pro Leu Val Ser Ile Ile Arg Asp Leu1085 1090 1095 Glu Lys Leu Ser Ser Tyr Leu Leu Asp Met Tyr Lys Val AspAla 1100 1105 1110 Gly Thr Gln Xaa Gln Leu Gln Ile Asp Ser Val Phe LysGly Ser 1115 1120 1125 Asn Leu Phe Val Ala Ala Pro Lys Thr Gly Asp IleSer Asp Met 1130 1135 1140 Gln Phe Tyr Tyr Asp Lys Cys Leu Pro Gly AsnSer Thr Met Met 1145 1150 1155 Asn Asn Phe Asp Ala Val Thr Met Arg LeuThr Asp Ile Ser Leu 1160 1165 1170 Asn Val Lys Asp Cys Ile Leu Asp MetSer Lys Ser Val Ala Ala 1175 1180 1185 Pro Lys Asp Gln Ile Lys Pro LeuIle Pro Met Val Arg Thr Ala 1190 1195 1200 Ala Glu Met Pro Arg Gln ThrGly Leu Leu Glu Asn Leu Val Ala 1205 1210 1215 Met Ile Lys Arg Asn PheAsn Ala Pro Glu Leu Ser Gly Ile Ile 1220 1225 1230 Asp Ile Glu Asn ThrAla Ser Leu Val Val Asp Lys Phe Phe Asp 1235 1240 1245 Ser Tyr Leu LeuLys Glu Lys Arg Lys Pro Asn Lys Asn Val Ser 1250 1255 1260 Leu Phe SerArg Glu Ser Leu Asn Arg Trp Leu Glu Lys Gln Glu 1265 1270 1275 Gln ValThr Ile Gly Gln Leu Ala Asp Phe Asp Phe Val Asp Leu 1280 1285 1290 ProAla Val Asp Gln Tyr Arg His Met Ile Lys Ala Gln Pro Lys 1295 1300 1305Gln Lys Leu Asp Thr Ser Ile Gln Thr Glu Tyr Pro Ala Leu Gln 1310 13151320 Thr Ile Val Tyr His Ser Lys Lys Ile Asn Ala Ile Phe Gly Pro 13251330 1335 Leu Phe Ser Glu Leu Thr Arg Gln Leu Leu Asp Ser Val Asp Ser1340 1345 1350 Ser Arg Phe Leu Phe Phe Thr Arg Lys Thr Pro Ala Gln IleGlu 1355 1360 1365 Asp Phe Phe Gly Asp Leu Asp Ser His Val Pro Met AspVal Leu 1370 1375 1380 Glu Leu Asp Ile Ser Lys Tyr Asp Lys Ser Gln AsnGlu Phe His 1385 1390 1395 Cys Ala Val Glu Tyr Glu Ile Trp Arg Arg LeuGly Phe Glu Asp 1400 1405 1410 Phe Leu Gly Glu Val Trp Lys Gln Gly HisArg Lys Thr Thr Leu 1415 1420 1425 Lys Asp Tyr Thr Ala Gly Ile Lys ThrCys Ile Trp Tyr Gln Arg 1430 1435 1440 Lys Ser Gly Asp Val Thr Thr PheIle Gly Asn Thr Val Ile Ile 1445 1450 1455 Ala Ala Cys Leu Ala Ser MetLeu Pro Met Glu Lys Ile Ile Lys 1460 1465 1470 Gly Ala Phe Cys Gly AspAsp Ser Leu Leu Tyr Phe Pro Lys Gly 1475 1480 1485 Cys Glu Phe Pro AspVal Gln His Ser Ala Asn Leu Met Trp Asn 1490 1495 1500 Phe Glu Ala LysLeu Phe Lys Lys Gln Tyr Gly Tyr Phe Cys Gly 1505 1510 1515 Arg Tyr ValIle His His Asp Arg Gly Cys Ile Val Tyr Tyr Asp 1520 1525 1530 Pro LeuLys Leu Ile Ser Lys Leu Gly Ala Lys His Ile Lys Asp 1535 1540 1545 TrpGlu His Leu Glu Glu Phe Arg Arg Ser Leu Cys Asp Val Ala 1550 1555 1560Val Ser Leu Asn Asn Cys Ala Tyr Tyr Thr Gln Leu Asp Asp Ala 1565 15701575 Val Trp Glu Val His Lys Thr Ala Pro Pro Gly Ser Phe Val Tyr 15801585 1590 Lys Ser Leu Val Lys Tyr Leu Ser Asp Lys Val Leu Phe Arg Ser1595 1600 1605 Leu Phe Ile Asp Gly Ser Ser Cys 1610 1615 9 9 PRT Alfalfamosaic virus PEPTIDE (1)..(9) 9 Ser Cys Ala Trp Tyr Asn Arg Val Lys 1 510 9 PRT Brome mosaic virus PEPTIDE (1)..(9) 10 His Cys Val Trp Phe GluAsp Ile Ser 1 5 11 9 PRT Citrus leaf rugose virus PEPTIDE (1)..(9) 11Ser Cys Ala Trp Leu Ser Ser Leu Arg 1 5 12 9 PRT cucumber mosaic virusPEPTIDE (1)..(9) 12 His Cys Ile Trp Phe Pro Ser Met Lys 1 5 13 9 PRTSunn-hemp mosaic virus PEPTIDE (1)..(9) 13 Phe Asn Val Tyr Phe Pro AsnAla Lys 1 5 14 10 PRT Tobacco mosaic virus PEPTIDE (1)..(10) 14 Ser ValAsn Tyr Trp Phe Pro Lys Met Arg 1 5 10 15 9 PRT Tobacco rattle virusPEPTIDE (1)..(9) 15 Val Glu Lys Gln Phe Met Asp Lys Cys 1 5 16 9 PRTTurnip vein-clearing virus PEPTIDE (1)..(9) 16 Leu Asn Phe Trp Phe ProLys Val Arg 1 5 17 16 PRT Tobacco mosaic virus PEPTIDE (1)..(16) 17 SerSer Val Asn Tyr Trp Phe Pro Lys Met Arg Ala Pro Glu Lys Ala 1 5 10 15 1816 PRT Tobacco mosaic virus PEPTIDE (1)..(16) 18 Gly Thr Val Asn Tyr TrpPhe Pro Glu Met Arg Val Ala Lys Arg Thr 1 5 10 15 19 16 PRT Tobaccomosaic virus PEPTIDE (1)..(16) 19 Gly Ser Val Asn Tyr Trp Phe Pro GluMet Arg Val Ala Lys Arg Thr 1 5 10 15 20 16 PRT Tobacco mosaic virusPEPTIDE (1)..(16) 20 Gly Ser Val Asn Tyr Trp Ala Pro Glu Met Arg Val AlaLys Arg Thr 1 5 10 15 21 16 PRT Tobacco mosaic virus PEPTIDE (1)..(16)21 Gly Ser Val Asn Tyr Trp Tyr Pro Glu Met Arg Val Ala Lys Arg Thr 1 510 15 22 37 DNA Tobacco mosaic virus misc_feature (1)..(37) PCR primer22 ctcatttcgg gagcccagta attgactgat gatgaat 37 23 33 DNA Tobacco mosaicvirus misc_feature (1)..(33) PCR primer 23 tttcgggata ccagtaattgactgatgatg aat 33 24 37 DNA Tobacco mosaic virus misc_feature (1)..(37)PCR primer 24 ccatgccatg gcgctcgaga tggcatacac acagaca 37 25 30 DNATobacco mosaic virus misc_feature (1)..(30) PCR primer 25 cccttgctcaccatttgtgt tcctgcatcg 30 26 30 DNA Plasmid pEGFP misc_feature (1)..(30)PCR primer 26 atgcaggaac acaaatggtg agcaagggcg 30 27 34 DNA PlasmidpEGFP misc_feature (1)..(34) PCR primer 27 ccatgccatg gctcgagttacttgtacagc tcgt 34

1. A method for decreasing the degradation rate of an engineered proteinof interest in a plant cell comprising constructing a vector comprisinga nucleic acid fragment from position 1 to position 3348 of SEQ ID NO:1fused to a nucleotide sequence encoding said protein of interest, saidvector expressible in said plant cell; and introducing and expressingsaid vector in said plant cell to form a fused protein; wherein thedegradation rate of said fused protein is less than the degradation rateof said engineered protein of interest in said plant cell or a plantcell of the same species.
 2. A method for decreasing the degradationrate of an engineered protein of interest in a plant cell comprisingconstructing a vector comprising a nucleic acid fragment from position 1to position 4831 of SEQ ID NO:3 fused to a nucleotide sequence encodingsaid protein of interest, said vector expressible in said plant cell;and introducing and expressing said vector in said plant cell to form afused protein; wherein the degradation rate of said fused protein isless than the degradation rate of said engineered protein of interest insaid plant cell or a plant cell of the same species.
 3. A method forincreasing the degradation rate of an engineered protein of interest ina plant cell comprising constructing a vector comprising a nucleic acidfragment from position 1 to position 3348 of SEQ ID NO:5 fused to anucleotide sequence encoding said protein of interest, said vectorexpressible in said plant cell; and introducing and expressing saidvector in said plant cell to form a fused protein; wherein thedegradation rate of said fused protein is less than the degradation rateof said engineered protein of interest in said plant cell or a plantcell of the same species.
 4. The method of claim 3, wherein nucleotidesat positions 1096-1098 of SEQ ID NO:5 encode alanine or tyrosine.
 5. Amethod for increasing the degradation rate of an engineered protein ofinterest in a plant cell comprising constructing a vector comprising anucleic acid fragment from position 1 to position 4831 of SEQ ID NO:7fused to a nucleotide sequence encoding said protein of interest, saidvector expressible in said plant cell; and introducing and expressingsaid vector in said plant cell to form a fused protein; wherein thedegradation rate of said fused protein is less than the degradation rateof said engineered protein of interest in said plant cell or a plantcell of the same species.
 6. The method of claim 5, wherein nucleotidesat positions 1096-1098 of SEQ ID NO:7 encode alanine or tyrosine.
 7. Themethod of claim 1, 2, 3, 4, 5 or 6, wherein said vector is integratedinto the genome of said plant cell.
 8. A plant cell transformedaccording to a method comprising constructing a vector comprising anucleic acid fragment fused to a nucleotide sequence encoding saidprotein of interest, said nucleic acid fragment selected from the groupconsisting of from position 1 to position 3348 of SEQ ID NO:1, fromposition 1 to position 4831 of SEQ ID NO:3, from position 1 to position3348 of SEQ ID NO:5, and from position 1 to position 4831 of SEQ IDNO:7, and said vector expressible in said plant cell; and introducingand expressing said vector in said plant cell to form a fused protein;wherein the degradation rate of said fused protein is less than thedegradation rate of said engineered protein of interest in said plantcell or a plant cell of the same species.
 9. A plant generated from theplant cell transformed according to a method comprising constructing avector comprising a nucleic acid fragment fused to a nucleotide sequenceencoding said protein of interest, said nucleic acid fragment selectedfrom the group consisting of from position 1 to position 3348 of SEQ IDNO:1, from position 1 to position 4831 of SEQ ID NO:3, from position 1to position 3348 of SEQ ID NO:5, and from position 1 to position 4831 ofSEQ ID NO:7, and said vector expressible in said plant cell; andintroducing and expressing said vector in said plant cell to form afused protein; wherein the degradation rate of said fused protein isless than the degradation rate of said engineered protein of interest insaid plant cell or a plant cell of the same species.
 10. A purifiednucleic acid comprising a nucleic acid fragment from position 1 toposition 3348 of SEQ ID NO:1 fused to a DNA sequence encoding a proteinof interest.
 11. The purified nucleic acid of claim 10, whereinexpression of said purified nucleic acid in a plant cell results in afusion protein having increased stability when compared to the stabilityof said protein of interest engineered without fusion to a nucleic acidfragment from position 1 to position 3348 of SEQ ID NO:1 expressed in aplant cell of the same species.
 12. A purified nucleic acid comprising anucleic acid fragment from position 1 to position 4831 of SEQ ID NO:3fused to a DNA sequence encoding a protein of interest.
 13. The purifiednucleic acid of claim 12, wherein expression of said purified nucleicacid in a plant cell results in a fusion protein having increasedstability when compared to the stability of said protein of interestengineered without fusion to a nucleic acid fragment from position 1 toposition 4831 of SEQ ID NO:3 expressed in a plant cell of the samespecies.
 14. A purified nucleic acid comprising a nucleic acid fragmentfrom position 1 to position 3348 of SEQ ID NO:5 fused to a DNA sequenceencoding a protein of interest.
 15. The purified nucleic acid of claim14, wherein expression of said purified nucleic acid in a plant cellresults in a fusion protein having increased stability when compared tothe stability of said protein of interest engineered without fusion to anucleic acid fragment from position 1 to position 3348 of SEQ ID NO:5expressed in a plant cell of the same species.
 16. The purified nucleicacid of claim 14, wherein expression of said purified nucleic acid in aplant cell results in a fusion protein having decreased stability whencompared to the stability of said protein of interest engineered withoutfusion to said nucleic acid fragment from position 1 to position 3348 ofSEQ ID NO:5 expressed in a plant cell of the same species.
 17. Thepurified nucleic acid of claim 14 or 16, wherein nucleotides atpositions 1096-1098 of SEQ ID NO:5 encode alanine or tyrosine.
 18. Apurified nucleic acid comprising a nucleic acid fragment from position 1to position 4831 of SEQ ID NO:7 fused to a DNA sequence encoding aprotein of interest.
 19. The purified nucleic acid of claim 18, whereinexpression of said purified nucleic acid in a plant cell results in afusion protein having increased stability when compared to the stabilityof said protein of interest engineered without fusion to a nucleic acidfragment from position 1 to position 4831 of SEQ ID NO:7 expressed in aplant cell of the same species.
 20. The purified nucleic acid of claim18, wherein expression of said purified nucleic acid in a plant cellresults in a fusion protein having decreased stability when compared tothe stability of said protein of interest engineered without fusion tosaid nucleic acid fragment from position 1 to position 4831 of SEQ IDNO:7 expressed in a plant cell of the same species.
 21. The purifiednucleic acid of claim 18 or 20, wherein nucleotides at positions1096-1098 of SEQ ID NO:7 encode alanine or tyrosine.
 22. A fusionprotein comprising SEQ ID NO:2 fused to an amino acid sequence ofinterest.
 23. The fusion protein of claim 22, wherein said fusionprotein has increased stability in a plant cell compared to said aminoacid sequence of interest in a plant cell of the same species.
 24. Afusion protein comprising SEQ ID NO:4 fused to an amino acid sequence ofinterest.
 25. The fusion protein of claim 24, wherein said fusionprotein has increased stability in a plant cell compared to said aminoacid sequence of interest in a plant cell of the same species.
 26. Afusion protein comprising SEQ ID NO:6 fused to an amino acid sequence ofinterest.
 27. The fusion protein of claim 26, wherein said fusionprotein has increased stability in a plant cell compared to said aminoacid sequence of interest in a plant cell of the same species.
 28. Thefusion protein of claim 26, wherein said fusion protein has decreasedstability in a plant cell compared to said amino acid sequence ofinterest in a plant cell of the same species.
 29. The fusion protein ofclaim 26 or 28, wherein the amino acid at position 366 of SEQ ID NO:6 isalanine or tyrosine.
 30. A fusion protein comprising SEQ ID NO:8 fusedto an amino acid sequence of interest.
 31. The fusion protein of claim30, wherein said fusion protein has increased stability in a plant cellcompared to said amino acid sequence of interest in a plant cell of thesame species.
 32. The fusion protein of claim 30, wherein said fusionprotein has decreased stability in a plant cell compared to said aminoacid sequence of interest in a plant cell of the same species.
 33. Thefusion protein of claim 30 or 32, wherein the amino acid at position 366of SEQ ID NO:8 is alanine or tyrosine.
 34. A vector purified nucleicacid encoding a fusion protein comprising SEQ ID NO:2 fused to an aminoacid sequence of interest.
 35. A vector comprising a purified nucleicacid encoding a fusion protein comprising SEQ ID NO:2 fused to an aminoacid sequence of interest.
 36. A plant cell transformed by a vectorcomprising a purified nucleic acid, said purified nucleic acid selectedfrom the group consisting of a purified nucleic acid encoding a fusionprotein comprising SEQ ID NO:2 fused to an amino acid sequence ofinterest, a purified nucleic acid encoding a fusion protein comprisingSEQ ID NO:4 fused to an amino acid sequence of interest; a purifiednucleic acid encoding a fusion protein comprising SEQ ID NO:6 fused toan amino acid sequence of interest; and a purified nucleic acid encodinga fusion protein comprising SEQ ID NO:8 fused to an amino acid sequenceof interest.
 37. A plant generated from a plant cell transformed by avector comprising a purified nucleic acid, said purified nucleic acidselected from the group consisting of a purified nucleic acid encoding afusion protein comprising SEQ ID NO:2 fused to an amino acid sequence ofinterest, a purified nucleic acid encoding a fusion protein comprisingSEQ ID NO:4 fused to an amino acid sequence of interest; a purifiednucleic acid encoding a fusion protein comprising SEQ ID NO:6 fused toan amino acid sequence of interest; and a purified nucleic acid encodinga fusion protein comprising SEQ ID NO:8 fused to an amino acid sequenceof interest.
 38. A method for decreasing the degradation rate of anengineered protein of interest in a plant cell comprising constructing avector comprising a nucleic acid sequence that encodes a membranebinding protein from the Sindbis-like plant virus family fused to anucleotide sequence encoding said protein of interest, said vectorexpressible in a plant cell; and introducing and expressing said vectorin said plant cell to form a fused protein; wherein the degradation rateof said fused protein is less than the degradation rate of saidengineered protein of interest in said plant cell or a plant cell of thesame species.
 39. The method of claim 38, wherein said membrane bindingprotein from the Sindbis-like plant virus family contains a “WFP” motifas depicted at amino acid position 365-367 of SEQ ID NO:2.
 40. A methodfor increasing the degradation rate of an engineered protein of interestin a plant cell comprising constructing a vector comprising a nucleicacid sequence that encodes a membrane binding protein from theSindbis-like plant virus family fused to a nucleotide sequence encodingsaid protein of interest, said vector expressible in a plant cell; andintroducing and expressing said vector in said plant cell to form afused protein; wherein the degradation rate of said fused protein isless than the degradation rate of said engineered protein of interest insaid plant cell or a plant cell of the same species.
 41. The method ofclaim 40, wherein said membrane binding protein from the Sindbis-likeplant virus family contains a mutation in the “WFP” motif as depicted atamino acid position 365-367 of SEQ ID NO:2.
 42. The method of claim 38or 40, wherein said vector is integrated into the genome of said plantcell.
 43. The method of claim 38, 39, 40 or 41, wherein the Sindbis-likeplant virus is selected from the group consisting of alfalfa mosaicvirus, brome mosaic virus, citrus leaf rugose virus, cucumber mosaicvirus, sunn-hemp mosaic virus, tobacco mosaic virus, tobacco rattlevirus, and turnip vein clearing virus.
 44. A plant cell transformedaccording to a method comprising constructing a vector comprising anucleic acid sequence that encodes a membrane binding protein from theSindbis-like plant virus family fused to a nucleotide sequence encodingsaid protein of interest, said vector expressible in a plant cell; andintroducing and expressing said vector in said plant cell to form afused protein; wherein the degradation rate of said fused protein isless than the degradation rate of said engineered protein of interest insaid plant cell or a plant cell of the same species.
 45. A plantgenerated from a plant cell transformed according to a method comprisingconstructing a vector comprising a nucleic acid sequence that encodes amembrane binding protein from the Sindbis-like plant virus family fusedto a nucleotide sequence encoding said protein of interest, said vectorexpressible in a plant cell; and introducing and expressing said vectorin said plant cell to form a fused protein; wherein the degradation rateof said fused protein is less than the degradation rate of saidengineered protein of interest in said plant cell or a plant cell of thesame species.
 46. A purified nucleic acid comprising a nucleic acidfragment encoding a membrane binding protein from the Sindbis-like plantvirus fused to a DNA sequence encoding a protein of interest.
 47. Apurified nucleic acid comprising a nucleic acid fragment encoding amembrane binding protein from the Sindbis-like plant virus containing amutation in the “WFP” motif as depicted at amino acid position 365-367of SEQ ID NO:2 fused to a DNA sequence encoding a protein of interest.48. The purified nucleic acid of claim 46 or 47, wherein theSindbis-like plant virus is selected from the group consisting ofalfalfa mosaic virus, brome mosaic virus, citrus leaf rugose virus,cucumber mosaic virus, sunn-hemp mosaic virus, tobacco mosaic virus,tobacco rattle virus, and turnip vein clearing virus.
 49. A fusionprotein comprising a membrane binding protein from the Sindbis-likeplant virus family fused to an amino acid sequence of interest.
 50. Afusion protein comprising a membrane binding protein from theSindbis-like plant virus family containing a mutation in the “WFP” motifas depicted at amino acid position 365-367 of SEQ ID NO:2 fused to anamino acid sequence of interest.
 51. The fusion protein of claim 49 or50, wherein said fusion protein has increased stability in a plant cellcompared to said amino acid sequence of interest in a plant cell of thesame species.
 52. The fusion protein of claim 49 or 50, wherein saidfusion protein has decreased stability in a plant cell compared to saidamino acid sequence of interest in a plant cell of the same species. 53.The fusion protein of claim 49 or 50, wherein the Sindbis-like plantvirus is selected from the group consisting of alfalfa mosaic virus,brome mosaic virus, citrus leaf rugose virus, cucumber mosaic virus,sunn-hemp mosaic virus, tobacco mosaic virus, tobacco rattle virus, andturnip vein clearing virus.
 54. A nucleic acid fragment encoding afusion protein comprising a membrane binding protein from theSindbis-like plant virus family fused to an amino acid sequence ofinterest.
 55. A vector comprising a nucleic acid fragment encoding afusion protein comprising a membrane binding protein from theSindbis-like plant virus family fused to an amino acid sequence ofinterest.
 56. A plant cell transformed with a vector comprising anucleic acid fragment encoding a fusion protein comprising a membranebinding protein from the Sindbis-like plant virus family fused to anamino acid sequence of interest.
 57. A plant generated from a plant celltransformed with a vector comprising a nucleic acid fragment encoding afusion protein comprising a membrane binding protein from theSindbis-like plant virus family fused to an amino acid sequence ofinterest.
 58. The plant cell of claim 8, wherein nucleotides atpositions 1096-1098 of SEQ ID NO:5 encode alanine or tyrosine.
 59. Theplant cell of claim 8, wherein nucleotides at positions 1096-1098 of SEQID NO:7 encode alanine or tyrosine.
 60. The plant cell of claim 8, 58 or59, wherein said vector is integrated into the genome of said plantcell.
 61. The plant of claim 9, wherein nucleotides at positions1096-1098 of SEQ ID NO:5 encode alanine or tyrosine.
 62. The plant ofclaim 9, wherein nucleotides at positions 1096-1098 of SEQ ID NO:7encode alanine or tyrosine.
 63. The plant of claim 9, 61 or 62, whereinsaid vector is integrated into the genome of said plant cell.
 64. Thevector purified nucleic acid of claim 34, wherein said fusion proteinhas increased stability in a plant cell compared to said amino acidsequence of interest in a plant cell of the same species.
 65. A vectorpurified nucleic acid encoding a fusion protein comprising SEQ ID NO:4fused to an amino acid sequence of interest.
 66. The vector purifiednucleic acid of claim 65, wherein said fusion protein has increasedstability in a plant cell compared to said amino acid sequence ofinterest in a plant cell of the same species.
 67. A vector purifiednucleic acid encoding a fusion protein comprising SEQ ID NO:6 fused toan amino acid sequence of interest.
 68. The vector purified nucleic acidof claim 67, wherein said fusion protein has increased stability in aplant cell compared to said amino acid sequence of interest in a plantcell of the same species.
 69. The vector purified nucleic acid of claim67, wherein said fusion protein has decreased stability in a plant cellcompared to said amino acid sequence of interest in a plant cell of thesame species.
 70. The vector purified nucleic acid of claim 67 or 69,wherein the amino acid at position 366 of SEQ ID NO:6 is alanine ortyrosine.
 71. A vector purified nucleic acid encoding a fusion proteincomprising SEQ ID NO:8 fused to an amino acid sequence of interest. 72.The vector purified nucleic acid of claim 71, wherein said fusionprotein has increased stability in a plant cell compared to said aminoacid sequence of interest in a plant cell of the same species.
 73. Thevector purified nucleic acid of claim 71, wherein said fusion proteinhas decreased stability in a plant cell compared to said amino acidsequence of interest in a plant cell of the same species.
 74. The vectorpurified nucleic acid of claim 71 or 73, wherein the amino acid atposition 366 of SEQ ID NO:8 is alanine or tyrosine.
 75. The vector ofclaim 35, wherein said fusion protein has increased stability in a plantcell compared to said amino acid sequence of interest in a plant cell ofthe same species.
 76. A vector comprising a purified nucleic acidencoding a fusion protein comprising SEQ ID NO:4 fused to an amino acidsequence of interest.
 77. The vector of claim 76, wherein said fusionprotein has increased stability in a plant cell compared to said aminoacid sequence of interest in a plant cell of the same species.
 78. Avector comprising a purified nucleic acid encoding a fusion proteincomprising SEQ ID NO:6 fused to an amino acid sequence of interest. 79.The vector of claim 78, wherein said fusion protein has increasedstability in a plant cell compared to said amino acid sequence ofinterest in a plant cell of the same species.
 80. The vector of claim78, wherein said fusion protein has decreased stability in a plant cellcompared to said amino acid sequence of interest in a plant cell of thesame species.
 81. The vector of claim 78 or 80, wherein the amino acidat position 366 of SEQ ID NO:6 is alanine or tyrosine.
 82. A vectorcomprising a purified nucleic acid encoding a fusion protein comprisingSEQ ID NO:8 fused to an amino acid sequence of interest.
 83. The vectorof claim 82, wherein said fusion protein has increased stability in aplant cell compared to said amino acid sequence of interest in a plantcell of the same species.
 84. The vector of claim 82, wherein saidfusion protein has decreased stability in a plant cell compared to saidamino acid sequence of interest in a plant cell of the same species. 85.The vector of claim 82 or 84, wherein the amino acid at position 366 ofSEQ ID NO:8 is alanine or tyrosine.
 86. The plant cell of claim 36,wherein said fusion protein has increased stability in a plant cellcompared to said amino acid sequence of interest in a plant cell of thesame species.
 87. The plant cell of claim 36, wherein said fusionprotein has decreased stability in a plant cell compared to said aminoacid sequence of interest in a plant cell of the same species.
 88. Theplant cell of claim 36, wherein the amino acid at position 366 of SEQ IDNO:6 is alanine or tyrosine.
 89. The plant cell of claim 36, wherein theamino acid at position 366 of SEQ ID NO:8 is alanine or tyrosine. 90.The plant of claim 37, wherein said fusion protein has increasedstability in a plant cell compared to said amino acid sequence ofinterest in a plant cell of the same species.
 91. The plant of claim 37,wherein said fusion protein has decreased stability in a plant cellcompared to said amino acid sequence of interest in a plant cell of thesame species.
 92. The plant of claim 37, wherein the amino acid atposition 366 of SEQ ID NO:6 is alanine or tyrosine.
 93. The plant ofclaim 36, wherein the amino acid at position 366 of SEQ ID NO:8 isalanine or tyrosine.
 94. The plant cell of claim 44, wherein saidmembrane binding protein from the Sindbis-like plant virus familycontains a mutation in the “WFP” motif as depicted at amino acidposition 365-367 of SEQ ID NO:2.
 95. The plant cell of claim 44, whereinsaid vector is integrated into the genome of said plant cell.
 96. Theplant cell of claim 94, wherein said vector is integrated into thegenome of said plant cell.
 97. The plant cell of claim 44, 94, 95 or 96,wherein the Sindbis-like plant virus is selected from the groupconsisting of alfalfa mosaic virus, brome mosaic virus, citrus leafrugose virus, cucumber mosaic virus, sunn-hemp mosaic virus, tobaccomosaic virus, tobacco rattle virus, and turnip vein clearing virus. 98.The plant of claim 45, wherein said membrane binding protein from theSindbis-like plant virus family contains a mutation in the “WFP” motifas depicted at amino acid position 365-367 of SEQ ID NO:2.
 99. The plantof claim 45, wherein said vector is integrated into the genome of saidplant cell.
 100. The plant of claim 98, wherein said vector isintegrated into the genome of said plant cell.
 101. The plant of claim45, 98, 99 or 100, wherein the Sindbis-like plant virus is selected fromthe group consisting of alfalfa mosaic virus, brome mosaic virus, citrusleaf rugose virus, cucumber mosaic virus, sunn-hemp mosaic virus,tobacco mosaic virus, tobacco rattle virus, and turnip vein clearingvirus.
 102. The fusion protein of claim 51, wherein the Sindbis-likeplant virus is selected from the group consisting of alfalfa mosaicvirus, brome mosaic virus, citrus leaf rugose virus, cucumber mosaicvirus, sunn-hemp mosaic virus, tobacco mosaic virus, tobacco rattlevirus, and turnip vein clearing virus.
 103. The fusion protein of claim52, wherein the Sindbis-like plant virus is selected from the groupconsisting of alfalfa mosaic virus, brome mosaic virus, citrus leafrugose virus, cucumber mosaic virus, sunn-hemp mosaic virus, tobaccomosaic virus, tobacco rattle virus, and turnip vein clearing virus. 104.A nucleic acid fragment encoding a fusion protein comprising a membranebinding protein from the Sindbis-like plant virus family containing amutation in the “WFP” motif as depicted at amino acid position 365-367of SEQ ID NO:2 fused to an amino acid sequence of interest.
 105. Thenucleic acid fragment of claim 54 or 104, wherein the Sindbis-like plantvirus is selected from the group consisting of alfalfa mosaic virus,brome mosaic virus, citrus leaf rugose virus, cucumber mosaic virus,sunn-hemp mosaic virus, tobacco mosaic virus, tobacco rattle virus, andturnip vein clearing virus.
 106. A vector comprising a nucleic acidfragment encoding a fusion protein comprising a membrane binding proteinfrom the Sindbis-like plant virus family containing a mutation in the“WFP” motif as depicted at amino acid position 365-367 of SEQ ID NO:2fused to an amino acid sequence of interest.
 107. The vector of claim 55or 106, wherein the Sindbis-like plant virus is selected from the groupconsisting of alfalfa mosaic virus, brome mosaic virus, citrus leafrugose virus, cucumber mosaic virus, sunn-hemp mosaic virus, tobaccomosaic virus, tobacco rattle virus, and turnip vein clearing virus. 108.A plant cell transformed with a vector comprising a nucleic acidfragment encoding a fusion protein comprising a membrane binding proteinfrom the Sindbis-like plant virus family containing a mutation in the“WFP” motif as depicted at amino acid position 365-367 of SEQ ID NO:2fused to an amino acid sequence of interest.
 109. The plant cell ofclaim 56 or 108, wherein the Sindbis-like plant virus is selected fromthe group consisting of alfalfa mosaic virus, brome mosaic virus, citrusleaf rugose virus, cucumber mosaic virus, sunn-hemp mosaic virus,tobacco mosaic virus, tobacco rattle virus, and turnip vein clearingvirus.
 110. A plant generated from a plant cell transformed with avector comprising a nucleic acid fragment encoding a fusion proteincomprising a membrane binding protein from the Sindbis-like plant virusfamily containing a mutation in the “WFP” motif as depicted at aminoacid position 365-367 of SEQ ID NO:2 fused to an amino acid sequence ofinterest.
 111. The plant cell of claim 57 or 110, wherein theSindbis-like plant virus is selected from the group consisting ofalfalfa mosaic virus, brome mosaic virus, citrus leaf rugose virus,cucumber mosaic virus, sunn-hemp mosaic virus, tobacco mosaic virus,tobacco rattle virus, and turnip vein clearing virus.